A molecularly annotated model of patient-derived colon cancer stem-like cells to assess genetic and non-genetic mechanisms of resistance to anti-EGFR therapy

Purpose Patient-derived xenografts (“ xenopatients ”) of colorectal cancer metastases have been essential to identify genetic determinants of resistance to the anti-EGF Receptor (EGFR) antibody cetuximab, and to explore new therapeutic strategies. From xenopatients, a genetically annotated collection of stem-like cultures (“ xenospheres ”) was generated and characterized for response to targeted therapies. Experimental Design. Xenospheres underwent exome-sequencing analysis, gene expression profile and in vitro targeted treatments to assess genetic, biological and pharmacological correspondence with xenopatients, and to investigate non-genetic biomarkers of therapeutic resistance. The outcome of EGFR family inhibition was tested in an NRG1 -expressing in vivo model. Results. Xenospheres faithfully retained the genetic make-up of their matched xenopatients over in vitro and in vivo passages. Frequent and rare genetic lesions triggering primary resistance to cetuximab through constitutive activation of the RAS signaling pathway were conserved, as well as the vulnerability to their respective targeted treatments. Xenospheres lacking such alterations (RAS wt ) were highly sensitive to cetuximab, but were protected by ligands activating the EGFR family, mostly NRG1. Upon reconstitution of NRG1 expression, xenospheres displayed increased tumorigenic potential in vivo , generated tumors completely resistant to cetuximab, and sensitive only to comprehensive EGFR family inhibition. Conclusions. Xenospheres are a reliable model to identify both genetic and non-genetic mechanisms of response and resistance to targeted therapies in colorectal cancer. In the absence of RAS pathway mutations, NRG1 and other EGFR ligands can play a major role in conferring primary cetuximab resistance, indicating that comprehensive inhibition of the EGFR family is required to achieve a significant therapeutic response.


Introduction
In metastatic colorectal cancer (mCRC), monoclonal antibodies against the Epidermal Growth Factor Receptor (EGFR), associated with standard chemotherapies, significantly extended the median overall survival (from 20 to 30 months) in a fraction of patients (1).In the vast majority of such responsive cases, EGFR inhibition is effective in the absence of any genetic alteration of the receptor (2,3).This exception to the "oncogene addiction" rule can be explained by the critical role that normal EGFR, physiologically stimulated by its ligand(s), plays in supporting colorectal cancer cell proliferation.This role is likely rooted in the cancer stem-like subpopulation, which, like its normal stem/progenitor counterpart, exquisitely depends on EGF signaling (4,5).In mCRC patients refractory to EGFR inhibition, the tumor often harbors genetic alterations such as KRAS or BRAF mutations, hijacking the control of the proliferative pathway downstream EGFR (6).Recently, new less frequent genetic mechanisms of resistance were identified, thanks to the analysis of a large collection of xenopatients.These mechanisms include ERBB2/MET amplifications (7,8), ERBB2 mutations (9), IGF2 overexpression (10) and EGFR mutations (11,12).Such alterations represent actionable therapeutic targets, and, indeed, patients with ERBB2 amplification underwent a successful clinical trial (13).
Interestingly, as shown by whole exome genetic analysis of an ample panel of xenopatients, in a fraction of refractory cases a genetic mechanism of resistance to EGFR inhibition has not been identified (11).In such cases, as suggested by previous studies (10,14) complete or partial resistance to cetuximab can be explained by the presence of autocrine or paracrine circuits of growth factors.Ligands of the EGF family, which fall in two main groups based on their ability to directly bind EGFR or another member of the EGFR family (15,16), can provide such a mechanism of resistance, as they could either compete or bypass cetuximab inhibition (17,18).For studying the outcome of paracrine signals, xenopatients may suffer from limitations, mainly because relevant growth factors can be underrepresented in the tumor microenvironment, or the murine ligands can fail to cross-react with human receptors.In a proof-of-concept study, we recently showed that xenopatient-derived cultures of stem-like cells (xenospheres) are a valuable system to address these issues in vitro and in vivo (5).
Xenospheres provide a unique counterpart of the original tumor and xenopatient, as they are amenable to in vitro quantitative measurement of proliferative responses, and, in vivo, they can regenerate phenocopies of the original tumor to provide a meaningful preclinical model (5,19).In this study we derived an ample collection of xenospheres and showed that they retain a remarkable genetic, biological and pharmacological correspondence with original mCRC xenopatients.In xenospheres lacking oncogenic activation of the RAS pathway, we could identify NRG1 as the main EGF family ligand able to substitute EGF in sustaining proliferation, and to induce primary resistance to cetuximab by engaging ERBB3.
We could then show that broad inhibition of the EGFR family is more effective than selective inhibition of EGFR alone.

Xenosphere generation and genetic characterization
All procedures involving animal experimentation were approved by the Italian Ministry of Health and by the internal Ethical Committee for Animal Experimentation.Xenospheres were derived as previously described (5).Genomic DNA was extracted with the Wizard® SV genomic DNA purification system (Promega) and analyzed for KRAS, NRAS, BRAF, PIK3CA mutations as previously described (8).Whole exome sequencing analysis was performed by PGD (Personal Genome Diagnostics) as previously described for the corresponding xenopatients, using the same settings and specific controls (11).Data concerning gene mutations and copy number variations (CNV) were extracted and analyzed.CNV include complete gene deletions and copy gains >4.

Flow-cytometric analysis
Immunophenotype was performed by incubating 2x10 5 cells with appropriate dilutions of the following antibodies: anti-EGFR-PE (BD Biosciences) anti-ERBB3-APC (BioLegend).DAPI was added for dead cell exclusion.Samples were analyzed in a CyAN ADP (DakoCytomation).Data were processed using Summit 4.3 software (DakoCytomation).

Generation of NRG1-expressing xenospheres
KRAS wt -Ctx-S xenospheres (CRC0059 and CRC0078) were stably transduced with a human NRG1-expressing lentiviral vector (Origene).NRG1 RNA expression was evaluated in vitro by qPCR using TaqMan® gene expression probe Hs00247620_m1 on ABI PRISM 7900HT sequence detection system according to manufacturer instructions.Ubiquitin (Hs00824723_m1) and beta-actin (Hs99999903_m1) were used as housekeeper genes.In vivo NRG1 RNA expression was evaluated by RNA-ISH using RNAscope ® technology (Advanced Cell Diagnostic, Inc.) according to manufacturer instructions.

Spheropatient generation and therapy
Dissociated xenospheres (5x10 4 cells) were resuspended in a 1:1 mixture of stem-cell medium and matrigel (BD Bioscience), and subcutaneously injected into NOD/SCID male mice (Charles River Laboratories).When tumors reached an average volume of 400 mm 3 , mice were randomized and treated with 20 mg/kg cetuximab (Merck) twice-weekly by IP injection, and/or with 150 mg/kg of lapatinib (Hospital Pharmacy), or 40 mg/kg of neratinib (Selleckchem), or 20 mg/kg of afatinib (Sequoia Research Products) by daily oral gavage.
Tumor size was measured once-weekly by caliper, and volume was calculated using the formula (4/3πd/2) 2 D/2, where d is the minor and D is the major tumor axis.

Statistical analysis
Results were expressed as means ± standard error of the mean (SEM).Statistical significance was evaluated by one-way ANOVA followed by Bonferroni's Multiple Comparison Test, using GraphPad Prism software.p<0.05 was considered statistically significant.

Derivation of a molecularly annotated xenosphere biobank
From mCRC xenopatients (8), we previously derived and characterized a small panel of "xenospheres" (5).These were shown to fulfill the requirements of colon cancer stem-like cells as, (i) in vitro, they retained self-renewal and pseudo-differentiative ability, and (ii) upon injection into immunocompromised mice, they formed tumors ("spheropatients") that reproduced both the same histological features and therapeutic response to cetuximab as the original tumors (Fig. 1A) (5).
The gene expression profiles of 28 randomly chosen matched xenospheres and xenopatients were compared to investigate the biological correspondence between the two models, showing a high level of overlapping between the transcriptome of matched samples (Fig. 1C and Supplementary Table-S2).Overall, these findings show that in vitro cultured colon cancer stem-like cells maintain both genetic and biological features of the tumor of origin.
Interestingly, the somatic mutations shared between xenospheres and the matched xenopatients displayed an average allelic frequency of ~50%, while lesions private to either model displayed a frequency of ~30% (Fig. 2D).This is in accordance with the notion that genetic alterations required for the oncogenic phenotype ("driver" lesions), should be both shared within the same cell population (high allelic frequency) and conserved during the passage from tumor tissue to xenosphere culture.These features are consistent with the neutral tumor evolution model, recently proposed also for colorectal cancer (23,24).Moreover, somatic mutations common to xenospheres and xenopatients displayed highly similar allelic frequencies in the two models, further attesting that xenospheres faithfully retain the genetic make-up of xenopatients (Fig. 2E).Exceptions were CRC0078 xenospheres, which displayed a lower grade of correspondence with the original xenopatient, both in the number of shared somatic mutations and their allelic frequency correlation (Fig. 2C,E).This could be explained by two mechanisms: (i) in vitro selection of a pre-existing xenopatient subclone (due to sample bias or to selection during cell culture) and/or (ii) xenosphere genetic drift occurred during multiple in vitro passages.To identify the underlying mechanism, WES analysis of CRC0078 xenospheres was longitudinally compared with that of two further in vitro derivatives of the same lineage: (i) a secondary CRC0078 xenosphere (CRC0078 2nd ), derived from the spheropatient, and previously shown to retain the same biological and tumorigenic properties of the primary xenosphere (5), and (ii) a primary cell culture selected in adherent standard cell culture conditions from the secondary xenosphere (CRC0078 ad ) (Fig. 3A).While the total number of CNV was conserved in the three in vitro models (Fig. 3B), a progressive increase of somatic mutations was observed in CRC0078 2nd and CRC0078 ad (Fig. 3C).As the number of mutations shared between each model with the original xenopatient remained constant, this increase indicated accumulation of new private lesions.Notably, the average allelic frequency of such lesions was lower, as compared with the frequency of lesions shared with the original xenopatient (Fig. 3D), as observed in all other xenospheres (Fig. 2D).Moreover, the allelic frequencies of single genes shared between each in vitro model and the xenopatient (Fig. 3E), or between the different in vitro models (Fig. 3F), were highly conserved.These data suggest that CRC0078 xenospheres likely derive from a pre-existing xenopatient subclone, and can accumulate new mutations that, however, for their low allelic frequency, are supposed to be mainly passengers.
Collectively, WES analyses indicate that xenospheres qualitatively and quantitatively reproduce, and stably retain over multiple in vitro passages, the landscape of relevant genetic alterations of the original tumors.

The cetuximab response of xenospheres correlates with genetic and non-genetic biomarkers of resistance.
Having shown that xenospheres faithfully retain both biological and genetic features of xenopatients, we assessed the response of xenospheres to anti-EGFR therapy with cetuximab, and its correlation with the presence of genetic determinants of resistance.The response was assessed in a panel of 46 xenospheres, using previously described cell culture conditions (5).
Interestingly, KRAS wt -Ctx-R xenospheres were enriched with cases harboring genetic alterations activating the RAS pathway (Fig. 4B).These lesions were recently described to be negative predictors of response to anti-EGFR therapy, and were exploited as new targets to overcome resistance in xenopatients (8)(9)(10)(11).Coherently, we observed similar correspondences in xenospheres (Fig. 4C): those harboring somatic mutations in the EGFR extracellular domain (CRC0104) and in ERBB2 (CRC0151) were resistant to cetuximab but sensitive to EGFR/ERBB2 small molecule kinase inhibitors; BRAF mutated xenospheres (CRC0480) were resistant to cetuximab but sensitive to BRAF inhibition, and were partially protected by EGF, consistently with the notion that EGFR activation protect against BRAF inhibition in CRC (Fig. 4C) (25).
According to the presence of these oncogenic drivers, KRAS wt -Ctx-R xenospheres were fully independent of exogenous EGF for their proliferation, similarly to KRAS mut xenospheres (Supplementary Fig. 1A).Vice-versa, the KRAS wt -Ctx-S subgroup mainly included xenospheres that lacked mutations known to activate the RAS pathway (Fig. 4B), and was strongly dependent on exogenous EGF for their proliferation (Supplementary Fig. 1A).This indicates that these xenospheres lack a cell-autonomous genetic driver of proliferation.Therefore they represent cases that, on the one hand, are susceptible to EGFR inhibition, and, on the other hand, can be affected by growth factors that can protect against different kinase inhibitors (26,27).Remarkably, the latter could be underestimated in conventional xenopatients, as we showed for HGF (5).
Therefore, we set out to systematically investigate which signals could confer to KRAS wt xenospheres the ability to circumvent the proliferative block imposed by EGFR inhibition with cetuximab.KRAS wt xenospheres were kept in the presence of growth factors that activate the EGFR family (the EGFR ligands EGF, TGFα, HBEGF, EREG, AREG and the ERBB3 ligand NRG1), and other tyrosine kinase receptors (bFGF and HGF).The response to cetuximab, measured as cell viability, was compared to that of untreated cells (Fig. 4D,E and Supplementary Fig. S1B).As expected, viability of the KRAS wt -Ctx-R xenosphere group kept in basal medium was overall poorly affected by cetuximab, and poorly modulated in the presence of the other growth factors (Fig. 4D).In contrast, the viability of the KRAS wt -Ctx-S xenosphere group was strongly impaired by cetuximab in basal medium, but it was at least in part preserved in the presence of other growth factors (Fig. 4E).These showed different abilities to protect from cetuximab: (i) negligible for EREG and AREG (viability < 0.4); (ii) intermediate for TGFα, HBEGF, HGF and bFGF (0.4 < viability < 0.8); (iii) maximum for EGF and NRG1 (viability > 0.8).
Taken together, these data indicate that xenospheres reproduce the correspondence observed in xenopatients between the genetic make-up on the one hand, and the therapeutic response to cetuximab or other targeted drugs on the other.Moreover, xenospheres lacking any known genetic determinants of resistance retain cetuximab sensitivity and can be exploited to qualitatively and quantitatively measure the ability of growth factors to compensate for EGFR inhibition.

EGF family ligands differently modulate EGFR activity and cetuximab response
In order to investigate the mechanisms of cetuximab primary resistance conferred by exogenous growth factors, we focused on the EGF family of ligands, whose expression in patients has been correlated with the cetuximab response (28)(29)(30).First, expression of EGFR family members ERBB1/2/3 (not ERBB4, undetectable in gene expression profiling) was assessed in western blots of xenospheres kept in basal medium (Supplementary Fig. S2A).
EGFR and ERBB3 proteins were expressed by most xenospheres, with the exception of one single case (CRC0729) that did not express EGFR and was completely insensitive to any of its ligands (Supplementary Fig. S2A,B).ERBB2 was significantly detectable in only two cases (CRC0394 and CRC0080), one of which (CRC0080) was ERBB2-amplified (Supplementary Fig. S2A).
Next we investigated whether the different ability of EGF family ligands to sustain cetuximab resistance (as shown in Fig. 4E) was associated with ligand-specific mechanisms of EGFR family activation.EGF-like ligands such as EGF, TGFα, HBEGF, AREG, and EREG, that bind EGFR (15), can directly outcompete the antibody, while NRG1, which binds ERBB3 and induces formation of heterodimers with either EGFR or ERBB2, is likely to bypass EGFR inhibition by cetuximab (Supplementary Fig. S3A).KRAS wt -Ctx-S xenospheres were kept in the presence of each ligand and biochemically analyzed.All EGFlike ligands activated ERK and AKT to a similar extent, while NRG1 hyperactivated both (Fig. 5A).The respective ability of EGF-like ligands to protect from cetuximab was correlated with a different ability to modulate EGFR total protein levels (Fig. 5A) and cellsurface expression (Fig. 5B): indeed, EGF (maximum protection against cetuximab) induced complete EGFR internalization and degradation, while TGFα and HBEGF (intermediate protection) showed only a partial effect on EGFR, and EREG and AREG (negligible protection) did not induce any EGFR internalization and degradation (Fig. 5A,B).As expected, NRG1 did not cause EGFR internalization, which mostly requires EGFR homodimerization (31,32).Interestingly, the EGF ability to induce EGFR internalization is inversely proportional to its concentration, and becomes negligible at doses that induce cell proliferation but are fully inhibited by cetuximab (Supplementary Fig. S3B,C).We can thus hypothesize that EGF-like ligands (or ligand concentrations) that do not induce efficient EGFR downregulation from the cell surface leave the receptor exposed to the competing activity of cetuximab, while those that induce internalization provide an escape from cetuximab and a site of intracellular signaling.

NRG1 sustains xenosphere resistance to cetuximab but full sensitivity to lapatinib
The above findings highlighted that NRG1 can powerfully activate EGFR signaling -the essential proliferative pathway in colorectal cancer stem cells -in a way alternative to EGF, which bypasses cetuximab inhibition.As the effect of NRG1 on colorectal cancer stem-like cells has been only preliminarily characterized (33), we investigated its ability to sustain xenosphere propagation.Six KRAS wt -Ctx-S xenospheres were long-term cultured either in the standard medium or in the corresponding medium where EGF was replaced by NRG1.
Beside a stronger ERK and AKT activation by NRG1, as compared with EGF (Supplementary Fig. S3D and data not shown), no significant differences were observed in the long-term proliferative potential (not shown) and, interestingly, even in the global gene expression profiles (Supplementary Fig. S4), suggesting that NRG1 could be interchangeable with EGF to support xenosphere expansion.
RNAseq analysis of xenopatients unveiled that, while most of EGF-like ligands are expressed by colorectal cancer cells, thus triggering autocrine loops, NRG1 is seldom expressed, similarly to HGF (20).However, while it is known that HGF is mainly secreted by cancer-associated fibroblasts (34), the source of NRG1 is still poorly understood, although it may include bone marrow-derived mesenchymal stem cells (35) or infiltrating lymphoid cells (36).Therefore, as in the case of HGF, the immunocompromised xeno-or spheropatient can be inadequate to evaluate NRG1 contribution to cetuximab resistance.
To circumvent this limitation, two KRAS wt -Ctx-S xenospheres were transduced with a lentiviral construct to induce autocrine expression of human NRG1 (Supplementary Fig. S5A,B).In a proliferation assay, NRG1-expressing xenospheres grew in the absence of either EGF or bFGF, showing complete resistance to cetuximab, but gaining full sensitivity to lapatinib (Fig. 5C).The same result was obtained in cells cultured in conventional cell-line conditions (i.e. in the presence of serum and adhesion, Supplementary Fig. S5C).This effect was specific as no inhibition was observed with an unrelated MET inhibitor (JNJ-38877605, Fig. 5D).Interestingly, the specific EGFR small molecule inhibitor gefitinib induced a weaker response as compared to lapatinib (Fig. 5D), suggesting that NRG1 exerts its effect through both ERBB3/EGFR and ERBB3/ERBB2 heterodimers.As in the case of stimulation with exogenous NRG1 (Supplementary Fig. S3D), NRG1-expressing xenospheres displayed constitutive ERBB3 phosphorylation, and a stronger activation of both ERK and AKT/S6 kinase pathways as compared with their parental counterparts (Fig. 5E).These pathways were inhibited by lapatinib but almost unaffected by cetuximab (Fig. 5E).EGFR was activated in both NRG1-expressing and parental xenospheres (for the presence of EGF in basal medium), but, whereas in parental xenospheres EGFR phosphorylation was completely abrogated by cetuximab, in NRG1-expressing xenospheres it was inhibited only by lapatinib (Fig. 5E).
In order to simulate paracrine secretion, a murine fibroblast primary culture (derived from a xenopatient) was transduced with human NRG1 (Supplementary Fig. S5E).
Representative KRAS wt -Ctx-S xenospheres were stimulated with conditioned media obtained from control and NRG1-expressing fibroblasts, and treated with either cetuximab or lapatinib.
Only the conditioned medium from NRG1-expressing fibroblasts could induce proliferation similarly to EGF, an effect inhibited by lapatinib but not by cetuximab (Supplementary Fig. S5E), as observed in the autocrine model (Fig. 5D).
Taken together, these experiments show that either autocrine or paracrine NRG1 powerfully stimulates proliferative EGFR signaling through ERBB3, and that this activity can be inhibited by the EGFR family kinase inhibitor lapatinib, but not by cetuximab.

NRG1-expressing KRAS wt xenospheres are highly tumorigenic, and generate tumors resistant to cetuximab but sensitive to pan-HER inhibitors.
In order to assess NRG1 ability to sustain cetuximab resistance in vivo, and explore ways to circumvent it, NRG1-expressing KRAS wt -Ctx-S xenospheres (CRC0078 and CRC0059) and their parental counterparts were injected into NOD/SCID mice (Fig. 6A).These xenospheres were previously shown to form spheropatients highly sensitive to cetuximab treatment (Supplementary Fig. S6A) (5).One month after injection, NRG1-expressing xenospheres formed tumors that reached volumes suitable for starting treatments (~500 mm 3 for CRC0078 and ~200 mm 3 for CRC0059), while no parental xenosphere had formed detectable tumors (Fig. 6B).These appeared later, and reached volumes comparable to those expressing NRG1 only after ~3 months (not shown), indicating that NRG1 strongly increases the xenosphere tumorigenic potential.RNA in-situ hybridization confirmed that NRG1 was highly expressed only in tumors derived from NRG1-expressing xenospheres (Fig. 6C).Spheropatients were then treated with cetuximab and/or lapatinib.CRC0078-NRG1 and CRC0059-NRG1 spheropatients were completely resistant to cetuxumab, as tumors grew with the same rate as controls (Fig. 6D).In contrast, the same tumors were arrested by lapatinib.No additive/synergistic effect was observed by lapatinib and cetuximab combination (Fig. 6D).
Similar results (growth arrest) were observed by treating CRC0078-NRG1 xenopatients with two other pan-HER inhibitors, afatinib or neratinib (Fig, 6E), which cause irreversible inhibition of the catalytic site, and were shown to be more potent than lapatinib in ERBB2amplified/mutated xenopatients (9,37).All three inhibitors effectively and comparably downregulated ERK and AKT signaling in tumors (Fig. 6F, Supplementary Fig. S6B).These results indicate that NRG1 can powerfully stimulate tumor growth in vivo, which can be arrested only by pan-HER inhibitors.

Discussion
mCRC xenopatients were fruitfully exploited by us and others as a translational platform to identify mechanisms of both primary and secondary resistance to cetuximab, and assess new targeting agents to bypass them (38).The identification of ERBB2 amplification as a mechanism of primary cetuximab resistance (8) has been recently translated into a successful clinical trial (13).
In vitro preclinical models are required as well to investigate both genetic and nongenetic mechanisms of response and resistance to conventional and targeted therapies.In colorectal cancer, different cell cultures approaches have been developed, including (i) conventional cell lines (39,40), organoids (41)(42)(43) and spheroids (19,(44)(45)(46), each displaying inherent and complementary advantages and limitations.
From mCRC xenopatients, we previously derived a small cohort of "xenospheres", showing that they retained cancer stem-like properties, and generated tumor xenografts ("spheropatients") that reproduced both the histotype and the pharmacological response to cetuximab of the corresponding xenopatients (5).Here we present an expanded cohort of 58 xenospheres, encompassing the main genetic lesions predictive of cetuximab resistance.By comparing gene expression profiles and WES analysis of a group of matched xenospheres and xenopatients, we observed a remarkable correspondence between the two models.WES analysis, in particular, showed that not only the type of genetic lesions, but also their respective allelic frequency, are passed on from xenopatients to xenospheres, indicating that xenospheres do not undergo a significant genetic drift from the original tumor.Moreover, they are also genetically stable over long-term propagation, as attested by longitudinal WES analysis of secondary xenospheres and a conventional cell line derived after extensive in vitro and in vivo passages of a prototypic case.Concerning their prerogatives as a reliable in vitro model, we can conclude that xenospheres display a faithful genetic correspondence with the original tumors.This genetic fidelity is shared with tumor organoids, whose derivation has been reported to be more efficient than xenospheres (90% vs. 45-50%) and whose growth, unlike that of xenospheres, tend to reproduce also cell pseudo-differentiation as in whole tumor tissues (41).However, with respect to organoids, xenospheres and, in general, cancer stem-like cells propagated in culture as spheroids, are endowed with the advantage to be more amenable to genetic manipulation and quantitative assessment of therapeutic outcomes at cancer stem cell level, either in vitro or after in vivo transplantation and tumor regeneration (5).
As previously shown by us in a limited number of cases (5), and recently observed also in tumor organoids (42), KRAS mut xenospheres display EGF-independent proliferative ability and, not surprisingly, are highly resistant to cetuximab in vitro.Here we show for the first time that also KRAS wt xenospheres derived from cetuximab-resistant xenopatients are mainly EGF-independent, and enriched with cases harboring genetic alterations such as EGFR extracellular domain mutations, ERBB2 mutation or amplification, BRAF mutations, PTEN deletion and IGF2 overexpression, each of which can provide a mechanistic explanation for cell-autonomous proliferation, and a therapeutic target (8)(9)(10)(11).
Conversely, we found that KRAS wt xenospheres derived from cetuximab-sensitive xenopatients (KRAS wt -Ctx-S) are devoid of genetic alterations able to hijack the proliferative pathway, and display strong EGF-dependent proliferative ability and in vitro cetuximab sensitivity.While representing patients that likely would benefit from anti-EGFR therapy, KRAS wt -Ctx-S xenospheres can also be susceptible to autocrine and paracrine signals that can counteract EGFR inhibition, as we previously showed for the HGF-MET axis (5).By screening the ability of a growth factors panel to induce primary resistance to cetuximab in vitro, we identified different responses that can explain pre-clinical and clinical observations.In particular, among the EGF family of ligands, EREG and AREG are completely unable to outcompete the antibody.Accordingly, their expression does not confer resistance, but, rather, is associated with cetuximab response both in patients (28,29) and in xenopatients (10).
Interestingly, unlike EGF, these ligands, although capable of inducing proliferation, cannot trigger EGFR internalization and degradation, suggesting that ligand-induced disappearance of EGFR from the cell surface is relevant to protect cells from cetuximab inhibition.
Similarly, TGFα and HBEGF induce only partial EGFR internalization and cetuximab resistance; this correlates with their enriched expression in KRAS wt xenopatients displaying either complete or partial resistance (disease stabilization) to cetuximab (10).
In our study, NRG1 emerged as a pivotal factor of primary resistance to EGFR inhibition.NRG1 is a ligand of the EGF family whose role in tumors has been poorly characterized.NRG1 binds the kinase-deficient ERBB3 receptor, and induces its heterodimerization with the other family members (EGFR, ERBB2 and ERBB4), which are required for ERBB3 transphosphorylation and the ensuing signaling activity (16).The mechanism of ERBB3 activation is complex and depends also on other EGF family ligands, specifically binding EGFR, as well as on dimerization with unrelated receptors such as the HGF receptor/MET (47).By this multiple interactions, ERBB3 is involved in primary and secondary resistance to EGFR or ERBB2 inhibition in several tumor types, including lung (47), head and neck (48), and ERBB2-amplified colorectal cancers (37).Concerning the specific role of NRG1, it was reported that this factor can induce in vitro resistance to various kinase inhibitors (26,33).In CRC, high levels of circulating NRG1 correlate with weak response to cetuximab (14).However, the activity of NRG1 has been so far poorly investigated at the mechanistic level in in vitro models.The ability of NRG1 to induce cetuximab resistance has been recently reported in a single conventional CRC cell line (49), which harbored a high-copy EGFR amplification, a genetic alteration uncommon in CRC.
Our study shows for the first time the protective ability of NRG1 in a large panel of mCRC stem-like cells that faithfully mirror the genetic make-up of the original tumors, thus achieving robust translational reliability.Moreover, we provide also the first evidence that NRG1 sustains long-term in vitro propagation of mCRC stem-like cells.
As NRG1 is produced by still elusive cells of the tumor microenvironment (20), to investigate its ability to sustain cetuximab resistance in vivo we induced NRG1 expression in KRAS wt -Ctx-S xenospheres, as we previously did for HGF (5).NRG1-expressing xenospheres displayed EGF-independent proliferative ability and full resistance to cetuximab.However, they were highly sensitive to lapatinib, a pan-HER inhibitor active in cancer cell lines harboring an NRG1-autocrine loop (50).Here we show for the first time that NRG1 dramatically increases the tumorigenic potential of xenospheres injected into NOD/SCID mice.In accordance with in vitro data, NRG1-expressing spheropatients displayed complete resistance to cetuximab treatment, but high sensitivity to different pan-HER inhibitors, including lapatinib, afatinib and neratinib.
It has been previously shown that rare genetic lesions in members of the EGFR family (EGFR and ERBB2 mutations, ERBB2 amplification) promote resistance to anti-EGFR therapy in KRAS wt xenopatients (8,9,11) and patients (13), which can be bypassed by different strategies that ultimately target multiple ERBBs.Here, by using a large cohort of colorectal cancer stem-like cultures, we show that the same strategy might also be effective in counteracting non-genetic mechanisms of primary resistance driven by ligands of the EGFR family, mainly NRG1.These results provide a proof-of-concept for the clinical investigation of NRG1 as a predictive biomarker of primary resistance to selective EGFR targeting, suggesting the requirement for broad inhibition of the entire EGFR family.This should apply primarily to RAS wt patients, and, possibly, to all patients lacking other biomarkers of resistance to EGFR inhibition, such as K/NRAS or BRAF mutations, MET amplification or IGF2 overexpression.As in the patient population NRG1 expression is likely a continuous variable, its clinical development as a predictive biomarker presents with the challenge to define a reliable threshold that discriminates positive from negative cases.As the cellular origin of NRG1 remains elusive, and likely involves tumor-associated cells, evaluation of insitu expression, although feasible either with immunohistochemistry or the more sensitive RNA in situ hybridization, can be significantly jeopardized by sample-bias.Integration of complementary approaches including measurements in circulating blood ( 14) should be recommended.Graphs represent tumor volume increase vs. day 0 ± s.e.m. (n = 6/group).Statistical analysis was performed using one-way ANOVA (p=0,004 for CRC0078; p<0,0001 for CRC0059).
Bonferroni Multiple Comparison Test was applied to compare treatments with vehicle to select those that showed detectable signal (detection P value = 0) in at least one samples.For each of such genes, only the probe with the highest variance of signal was selected.Pearson's correlations were performed for any possible PDX/Xenosphere permutation.The dataset was uploaded on the GEO Database (GEO accession number GSE101792).

Generation of NRG1-expressing xenopheres
To generate stable xenospheres (M016 and M049) or murine fibroblasts expressing NRG1, cells were transduced with a lentiviral vector generated by transfecting 293-T cells with different plasmids: the packaging construct encodes the HIV-1 Gag and Pol precursors, the regulatory proteins Tat and Rev, pMDL, the VSV-G expressing construct, and the transfer construct OriGene's TrueORF clone RC220134L1V (Origene).The ORF cloned in this vector was expressed as a tagged protein with c-terminal Myc-DDK tags.

Real time RT-PCR
Total RNA was extracted using miRNAeasy mini Kit according to manufacturer's instructions (Qiagen).1 µg of purified RNA was used as a template for cDNA synthesis with random and hexamer primers and high capacity reverse transcription kit (Applied Biosystem).To evaluate NGR1 expression Real-time PCR was performed using TaqMan® Universal Master Mix (Life Technologies) containing 200 ng of cDNA and a TaqMan® gene expression probe Hs00247620_m1 on ABI PRISM 7900HT sequence detection system.Relative quantification value for NGR1 gene expression was obtained by normalizing to ubiquitin and beta actin as endogenous controls.All samples were run in triplicate and the mean and standard deviation calculated.

Murine Fibroblast Conditioned Medium (mFIBR CM)
A murine fibroblast culture derived from a xenopatient was isolated and transduced to express human NRG1 as described above.Cells were plated in adhesive dishes in DMEM supplemented with 2 mM glutamine, penicillin-streptomycin, and 10% FBS.To obtain mFIBR CM, fibroblasts were grown up to confluence and then kept for 24h in basal stem-cell medium.

Figure 1 .
Figure 1.Derivation of a molecularly annotated xenospheres biobank.A, Xenospheres were derived from tumor samples of patients with metastatic colorectal cancers previously implanted into immunocompromised mice and expanded as patientderived xenografts (xenopatients).Xenosphere transplantation into immunocompromised mice (spheropatient) regenerates phenocopies of original tumors.B, Pie chart representing the relative distribution of genetic lesions in the total panel of 58 xenospheres (left) and in the 32 quadruple WT xenospheres (right).The number of cases harboring each genetic alteration is indicated.WT: wild-type; mut: mutation; high: overexpression; ampl: amplification; ecd: mutation in the extracellular domain; del: deletion.C, Heatmap representing the Pearson correlation of gene expression profiles of 28 matched xenospheres and xenopatients (GEO accession number GSE101792).

Figure 2 .
Figure 2. Whole exome sequencing analysis (WES) unveils faithful genetic correspondence between xenospheres and original xenopatients.A, Schematic representation of WES analysis comparison between xenospheres and xenopatients.B, C, Histograms representing the total number of copy number variations (CNV, B) and somatic mutations (C) observed in xenospheres (blue columns) and xenopatients (red columns), and those shared between them (green columns).D, Histogram representing the average allelic frequencies of somatic mutations either private or shared in xenospheres (blue/light blue columns) and xenopatients (red/orange columns).E, Graph representing the correlated allelic frequency of somatic mutations shared between xenospheres and xenopatients.Each dot represents a gene mutation.Pearson correlation between allelic frequencies in xenospheres vs. xenopatients is indicated.

Figure 3 .
Figure 3. Longitudinal WES analysis of different CRC0078 xenospheres unveils in vitro genetic stability.A, Schematic representation of the derivation of secondary CRC0078 2nd xenospheres, and CRC0078 Ad xenosphere-derived adherent cultures.B, C, Histogram representing the total number of copy number variations (CNV, B) and somatic mutations (C) observed in CRC0078 xenospheres, CRC0078 2nd xenospheres, and CRC0078 Ad cultures (blue columns), as compared with the original CRC0078 xenopatient (red columns), and the number of alterations shared between each in vitro model and the xenopatient (green columns).D,

Figure 4 .
Figure 4.The xenosphere response to cetuximab correlates with genetic and non-genetic biomarkers of resistance.A, Waterflow plot of the in vitro cetuximab response of a panel of 46 xenospheres.Cell viability was measured in xenospheres kept for 4 days in basal medium (with 0,4 ng/ml of EGF) and treated with cetuximab (10 μg/ml) or vehicle.Columns represent relative viability of xenospheres treated with cetuximab vs. vehicle.Relative viability > 0.5 (dotted line) indicates resistance, < 0.5 indicates sensitivity.Colors indicate the mutational status, as indicated in the legend.B, Pie charts of the genetic classification of KRAS wt xenospheres (n = 30), which are subdivided in two groups according to the annotation of the cetuximab response of the original xenopatients.Left: group derived from cetuximab-resistant (Ctx-R, n = 12) xenopatients.Right: group derived from cetuximab-sensitive xenopatients (Ctx-S, n = 18).C, Cell viability assay of CRC0104, CRC0480 and CRC0151 xenospheres kept for 4 days in stem-cell medium without any growth factor (NoGF) or with 0,4 ng/ml of EGF (Basal Medium), and treated as indicated (cetuximab 10 μg/ml, panitumumab 10 μg/ml, gefitinib 0,5 μM, Lapatinib 0,5 μM, PLX4720 2 μM).Columns represent the relative viability vs. vehicle (No GF) ± s.e.m (n = 3).D, E, Cell viability assay of KRAS wt Ctx-R (n=12, C) or KRAS wt Ctx-S (n=18, D) xenospheres kept for 4 days in basal medium alone (with 0,4 ng/ml of EGF), or supplemented with 20 ng/ml of the indicated growth factors, and treated with cetuximab (10 μg/ml) or vehicle.Dots represent the ratio of raw viability data between cetuximab-treated and vehicle-treated xenospheres.Each dot represents one single xenosphere.Red line: mean value for the group.Statistical analysis of the assay was performed with one-way ANOVA (p<0,0001).Bonferroni Multiple Comparison Test was applied to compare each growth factor with basal medium (**p<0,01; ***p<0,001).

Figure 6 .
Figure 6.NRG1-expressing KRAS wt -Ctx-S spheropatients are highly tumorigenic, and generate tumors resistant to cetuximab but sensitive to pan-HER inhibitors.A, Schematic representation of the generation of spheropatients by KRAS wt -Ctx-S and NRG1expressing KRAS wt -Ctx-S xenospheres.B, Graph representing the distribution of CRC0059 and CRC0078 parental or NRG1 expressing spheropatient tumor volumes one month after xenosphere injection (5 × 10 4 cells/mouse).Red line: Mean Value.Statistical analysis was performed using one-way ANOVA (p<0,0001).C, Micrographs of NRG1 RNA in Situ Hybridization (ISH) performed on histological tumor sections derived from CRC0059 and CRC0078 parental or NRG1 expressing spheropatients.Magnification: 20Χ.D, Tumor growth curves of CRC0059-NRG1 and CRC0078-NRG1 spheropatients treated as indicated.