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Ex vivo organotypic cultures for synergistic therapy prioritization identify patient-specific responses to combined MEK and Src inhibition in colorectal cancer

Abstract

Translating preclinical studies to effective treatment protocols and identifying specific therapeutic responses in individuals with cancer is challenging. This may arise due to the complex genetic makeup of tumor cells and the impact of their multifaceted tumor microenvironment on drug response. To find new clinically relevant drug combinations for colorectal cancer (CRC), we prioritized the top five synergistic combinations from a large in vitro screen for ex vivo testing on 29 freshly resected human CRC tumors and found that only the combination of mitogen-activated protein kinase kinase (MEK) and proto-oncogene tyrosine-protein kinase Src (Src) inhibition was effective when tested ex vivo. Pretreatment phosphorylated Src (pSrc) was identified as a predictive biomarker for MEK and Src inhibition only in the absence of KRASG12 mutations. Overall, we demonstrate the potential of using ex vivo platforms to identify drug combinations and discover MEK and Src dual inhibition as an effective drug combination in a predefined subset of individuals with CRC.

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Fig. 1: Optimizing conditions for robust and versatile EVOC.
Fig. 2: EVOC can predict the response of PDX tumors to treatment.
Fig. 3: In vitro screens of drug combinations identify multiple synergistic pairs in CRC cell lines.
Fig. 4: Ex vivo screening identifies the dual inhibition of MEK and Src as effective in a subset of human CRCs.
Fig. 5: High pSrc and inhibition of phospho-extracellular signal-regulated kinase (pERK) by Src inhibitor treatment are both predictive biomarkers of response to MEK/Src inhibition.
Fig. 6: Feedback phosphorylation of MEK as a result of MEK inhibition is abrogated by the addition of Src inhibitor in CRC tumors that are sensitive to MEK/Src inhibition.

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Data availability

Source data for the in vitro screens are provided as Supplementary Tables 3 and 4. Results of the evaluations of all the human samples for each drug and drug combination have been provided in Supplementary Table 6. Clinical data of the participants included in the study are provided in Supplementary Table 5. Mutations found in participant samples are provided in Supplementary Table 7. Drug doses used in the EVOC experiments are listed in Supplementary Table 2. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank all the members of the Straussman Lab for helpful discussions. We thank A.L. Barda, T. Oren, A. Itzhaki-Alfia and the Tel Aviv Sourasky Medical Center Biobank for their help with tissue recruitment. We thank E. Pikarsky for his willingness to review numerous pathology results in the early and critical stages of this work. This work was supported by a grant from the Rising Tide Foundation and by a research grant from the Fabricant-Morse Families Research Fund for Humanity. R.S. is the incumbent of the Roel C. Buck Career Development Chair.

Author information

Authors and Affiliations

Authors

Contributions

N.G., Y.Z., O.M. and R.S. conceptualized and designed the study. N.G. and R.S. developed the methodology. N.G., Y.Z., R. Weiser, A.J.B., S.H., O.J., E.S., A.G., D.S., I.B., I.K., J.B., G.P., E.B.-A., S.G., E.R., A.N., R. Weitzen, H.N., T.G., B.B., A.N. and G.L. acquired data (providing animals acquired and managed participants, provided facilities and so on). N.G., R.S., O.S., O.M., O.G., G.M., D.R. and D.H. analyzed and interpreted the data (for example, statistical analyses, biostatistics and computational analyses). N.G., G.M., Y.Z., A.G., S.G., R. Weitzen, A.N., T.G., G.L., H.N., B.B. and R.S. wrote, reviewed and/or revised the manuscript. R.S. supervised the study.

Corresponding author

Correspondence to Ravid Straussman.

Ethics declarations

Competing interests

R.S. and N.G. are founders, stockholders and paid consultants of the company CuResponse, which currently validates EVOC as a predictive biomarker for the effect of anticancer drugs. G.M. worked as a part-time pathologist for Curesponse. E.S. works as a part-time technician at CuResponse. No other potential conflicts of interest were disclosed by other authors.

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Nature Cancer thanks John Minna, Nicola Valeri and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Optimizing culture conditions for EVOC.

a, An example of H&E staining of colorectal cancer from a patient (top row), ovarian cancer tissue (middle row) and breast cancer tissue (bottom row) from PDX mouse models cultured in different media for five days. Black boxes mark the regions that were selected for magnification on the right. Representative pictures are presented from n=2 tumors for each of ovarian and breast cancer PDX models, and n=3 tumors for CRC cancer patients. Scale bar 50μM in low magnifications and 20μM in high magnifications. b, Examples of H&E staining of histological slices of ovarian PDX tumors used to determine the viability of cancer cells after five days in culture under the different conditions as labeled. A scoring system was used to categorize the percentage of viable cancer cells: 0 for less than 10% viable cancer cells, 1 for 10–30%, 2 for 31–60%, 3 for 61–90%, and 4 for more than 90%. c, Upper panel summarizes experiments performed using 5 human CRC tissues, in which H&E staining was used to determine the effect of using a grid or high oxygen partial pressure on the viability of cancer cells after five days in culture. The bottom panel shows representative histological slices from two of the tumors in the upper panel. Each experiment was performed using at least two tissue slices for each condition. Scale bar 40μM. d, H&E staining of a human non-small cell lung cancer (NSCLC) tumor that was sliced and immediately fixed or cultured on a titanium grid in 80% O2PP for 4 or 6 days. Scale bar 50μM. O2PP, oxygen partial pressure; DMSO, dimethyl sulfoxide.

Source data

Extended Data Fig. 2 EVOC can predict the response of PDX tumors to treatment.

a, Table summarizing the ex vivo (EV) vs. in vivo (IV) response to drugs across 16 models of colon, breast, and lung PDX tumors treated with different drugs. Ex vivo experiments were scored as follows: NR - no response (viability score 4); PR - partial response (viability scores 1–3); CR - complete response (viability score 0). In vivo experiments were scored by the percent reduction in fold change of the tumor growth between the treated and untreated control mice groups as follows: NR - No response (<30% reduction); PR - partial response (>=30 to <100% reduction); CR - complete or near-complete response (>=100% reduction). b,d,f, left panels, Bar plots demonstrating the viability scores of two NSCLC PDX tumors treated ex vivo for 4 days with a range of concentrations of docetaxel, cisplatin or trametinib. Bar errors represent the standard deviation of two (n=2) or three (n=3) tumors, each condition within an experiment was evaluated in at least two slices of tissue. Right panel, growth curves representing the in vivo response of tumors to docetaxel, cisplatin or trametinib, or vehicle control. In vivo data was generated by the Jackson Laboratories. Error bars represent standard error of the mean (for TM00356: n=8 for cisplatin and trametinib treatment and n=7 mice for vehicle and trametinib treatment; for TM00123: n=6 mice for vehicle and cisplatin treatment, n=4 mice for docetaxel treatment and n= 5 mice for trametinib treatment). c,e,g, Representative histological sections of tissues from (b), (d) and (f) stained with H&E. h,j, Left panel, bar plots demonstrating the viability scores of two breast cancer PDX tumors treated ex vivo for 4 days with a range of concentrations of 4-hydroxycyclophosphamide (active metabolite of the pro-drug cyclophosphamide) and docetaxel. h,j, Right panel, growth curves representing the in vivo response of tumors to cyclophosphamide, docetaxel, or vehicle control. In vivo data was generated by the Jackson Laboratories. Error bars represent standard error of the mean (for TM00096: n=9 mice for docetaxel treatment, n=8 mice for vehicle and n=7 mice for cyclophosphamide treatment; for TM00098: n=10 mice for cyclophosphamide and docetaxel treatment and n=9 mice for vehicle treatment). i,k, Representative histological sections of tissues from (h) and (j) stained with H&E. The p values were calculated using a two-sided rank test, comparing the viability scores of all drug concentrations tested across all experiments. Scale bars 100μM for all micrographs in the figure. Doc, docetaxel; Cis, cisplatin; Tra, trametinib; Cyc, cyclophosphamide, DMSO, dimethyl sulfoxide.

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Extended Data Fig. 3 Graphs depicting results from in vivo screens.

a, List of the 24 drugs tested and their primary targets. b, 276 drug combinations were tested on eight colorectal cancer cell lines (figure 3a - screen 2). Numbers represent the number of cell lines for which the combination of drugs was synergistic as determined by a Bliss score < 0.6 (Supplementary Table 4). c, Table listing the seven top synergistic drug combinations based on screen 3. For each drug pair, the table shows the number of cell lines in which the pair was synergistic (Bliss score <0.6) in >2 of the nine tested combinations. The total number of occurrences in which the bliss score was < 0.6 out of the possible 72 occurrences (9 drug concentrations X 8 cell lines) is also depicted. H-89 2HCl, H-89-dihydrochloride.

Source data

Extended Data Fig. 4 Examples of calibrating drug doses for ex vivo organ culture using CRC PDX tumors.

a,b,c, Representative IHC and H&E staining of tumors from the CRC PDX model TM00170 (a,b) or TM00164 (c) cultured ex vivo with increasing concentrations of different drugs. Tissues were fixed after 24–96 hours of treatment, as indicated in the figure. Viability scores are depicted in the right lower corners of the H&E stained slides. Scale bars 50μM (a,b) 40μM (c). At least two tissue slices were evaluated for each condition. PKA, protein kinase A; MDM2, Mouse double minute 2 homolog; H-89 2HCl, H-89 dihydrochloride; h, hour; DMSO, dimethyl sulfoxide.

Extended Data Fig. 5 The response of CRC human tumors to five combinatory treatments.

All tumors were treated ex vivo for 4 days with either DMSO control, or drugs as indicated. Viability of the tissue slices as reflected in H&E staining was scored from 0 to 4, by a pathologist blinded to the treatments: 0 for less than 10% viable cancer cells, 1 for 10–30%, 2 for 31–60%, 3 for 61–90%, and 4 for more than 90%. FOLFOX, 5-fluorouracil/oxaliplatin; FOLFIRI, 5-fluorouracil/irinotecan; DMSO, dimethyl sulfoxide.

Source data

Extended Data Fig. 6 The effect of MEK/Src inhibition on the proliferation of cancer cells.

Example of CRC tumor from patient 28 - a responder to dual inhibition of MEK and Src. Drugs (or DMSO control) were added 16–20 hours after the slicing of the tumor, and tissue slices were fixed after four complete days of treatment. Bromodeoxyuridine (BrdU) was added to all slices 18 hours before the end of the experiment. In each condition, at least two tissue slices were evaluated. a, Representative histological sections stained with H&E. b, Representative histological slices from the same EVOC slices in (a), stained for anti-BrdU. Note that proliferation is evident as measured by the incorporation of BrdU when the tissue is treated with either selumetinib or bosutinib, but is markedly reduced when treated with both drugs. Scale bar 50μM for all micrographs in the figure.

Extended Data Fig. 7 The Src inhibitors dasatinib and bosutinib and the MEK inhibitors trametinib and selumetinib show similar response when combined with MEK inhibition and tested ex vivo on human CRC.

Drugs (or DMSO control) were added 16–20 hours after the slicing of the tumor, and tissue slices were fixed after four complete days of treatment. In each condition, at least two tissue slices were evaluated. a,b, The effect of dasatinib combined with selumetinib is compared to the effect of bosutinib combined with selumetinib c, The effect of trametinib combined with dasatinib is compared to the combination of selumetinib and bosutinib. Viability scores are depicted in the right lower corners. Scale bar 20μM for all micrographs in the figure. 5-FU, 5-fluorouracil; DMSO, dimethyl sulfoxide.

Extended Data Fig. 8 Additional examples of the response of human CRC tumors to several drug combinations tested in EVOC.

a,b,c, Drugs (or DMSO control) were added 16–20 hours after the slicing of the tumor, and tissue slices were fixed after four complete days of treatment. In each condition, at least two tissue slices were evaluated. Viability scores are depicted in the right lower corners. Scale bar 40μM for all micrographs in the figure. 5-FU, 5-fluorouracil; Ox, oxaliplatin; Ir, irinotecan; H-89 2HCl, H-89 dihydrochloride; DMSO, dimethyl sulfoxide.

Extended Data Fig. 9 Ex vivo response to chemotherapy correlates with the clinical course in selected patients.

From the 28 patients that entered the study to test the response of their CRC tissue to chemotherapy and targeted dual therapy, four patients were available for evaluation and comparison to ex vivo data. Clinical evaluation of disease state was determined by PET/CT and interpreted by the treating physician independently and without knowledge of the results of the ex vivo study. In patients: progressive disease (PD) refers to an increase in tumor size, number or location, and can include an increase in tumor marker values and a worsening of the patients’ overall condition; partial response (PR) refers to a reduction in tumor size or number; stable disease (SD) refers to no change in tumor size or number. No response (NR) in EVOC refers to tissue viability score after treatment of 4 (more than 90% viable - see also Supplementary Table 6). Partial response (PR) in EVOC refers to tissue viability score from 1–2 (more than 10 but less than 60% - see also Supplementary Table 6). FOLFOX, 5-fluorouracil and oxaliplatin; FOLFIRI, 5-fluorouracil and irinotecan; MMC, mitomycin C; LAR, low anterior resection; mets, metastasis; AR, anterior resection; CA, carcinoma; CRC, colorectal carcinoma; CET, cetuximab; ERB, erlotinib; Tx, treatment; PR, partial response (green arrows); SD, stable disease (grey arrows); PD, progressive disease (black arrows). Light blue arrows indicate time of diagnosis. Royal blue arrows indicate operative intervention. Yellow arrows indicate recurrence of disease. Red arrows indicate when tissue was obtained for ex vivo organ culture.

Extended Data Fig. 10 Sensitivity to MEK and Src inhibition does not correlate with phosphorylation of Src in CRC cell lines.

The eight CRC cell lines used in the in vitro screens (see Fig. 3) were seeded in 6 cm plates and treated as labeled for 24 hours and then collected for western blot evaluation. To determine the best drug dose for each cell line we utilized the extensive data from the in vitro screens (see text and Fig. 3): The IC20 was determined in the in vitro screen #1 and the most synergistic dose of the combined MEK/Src inhibition (selumetinib/bosutinib) (either 0.5xIC20, IC20 or 2XIC20) was determined in the in vitro screen #3. We used the most effective synergistic dose for each cell line. a, Western blot analysis of eight CRC cell lines as labeled. b, Phosphorylated Src signal intensity determined as a ratio of HSP90 (heat shock protein 90) signal intensity used as loading control. Lines represent average intensity for each group and p value = 0.21 as determined by two-tailed student t-test. The experiment was performed in 8 different cell lines (n=8 cell lines; n=1 biological repeat). MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; Src, proto-oncogene tyrosine-protein kinase Src.

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Gavert, N., Zwang, Y., Weiser, R. et al. Ex vivo organotypic cultures for synergistic therapy prioritization identify patient-specific responses to combined MEK and Src inhibition in colorectal cancer. Nat Cancer 3, 219–231 (2022). https://doi.org/10.1038/s43018-021-00325-2

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