A clinical evaluation of an ex vivo organ culture system to predict patient response to cancer therapy.

Introduction: Ex vivo organ cultures (EVOC) were recently optimized to sustain cancer tissue for 5 days with its complete microenvironment. We examined the ability of an EVOC platform to predict patient response to cancer therapy. Methods: A multicenter, prospective, single-arm observational trial. Samples were obtained from patients with newly diagnosed bladder cancer who underwent transurethral resection of bladder tumor and from core needle biopsies of patients with metastatic cancer. The tumors were cut into 250 µM slices and cultured within 24 h, then incubated for 96 h with vehicle or intended to treat drug. The cultures were then fixed and stained to analyze their morphology and cell viability. Each EVOC was given a score based on cell viability, level of damage, and Ki67 proliferation, and the scores were correlated with the patients' clinical response assessed by pathology or Response Evaluation Criteria in Solid Tumors (RECIST). Results: The cancer tissue and microenvironment, including endothelial and immune cells, were preserved at high viability with continued cell division for 5 days, demonstrating active cell signaling dynamics. A total of 34 cancer samples were tested by the platform and were correlated with clinical results. A higher EVOC score was correlated with better clinical response. The EVOC system showed a predictive specificity of 77.7% (7/9, 95% CI 0.4-0.97) and a sensitivity of 96% (24/25, 95% CI 0.80-0.99). Conclusion: EVOC cultured for 5 days showed high sensitivity and specificity for predicting clinical response to therapy among patients with muscle-invasive bladder cancer and other solid tumors.

An Increase in Mucin2 Expression Is Required for Colon Cancer Progression Mediated by L1.

An induction in the expression of the cell adhesion receptor L1, a Wnt target gene, is a characteristic feature of Wnt/ß-catenin activation in colon cancer cells at later stages of the disease. We investigated the proteins secreted following L1 expression in colon cancer cells and identified Mucin2 among the most abundant secreted proteins. We found that suppressing Mucin2 expression in L1-expressing colon cancer cells inhibits cell proliferation, motility, tumorigenesis, and liver metastasis. We detected several signaling pathways involved in Mucin2 induction in L1-expressing cells. In human colon cancer tissue, Mucin2 expression was significantly reduced or lost in the adenocarcinoma tissue, while in the mucinous subtype of colon cancer tissue, Mucin2 expression was increased. An increased signature of L1/Mucin2 expression reduced the survival rate of human colon cancer patients. Thus, induction of Mucin2 expression by L1 is required during mucinous colon cancer progression and can serve as a marker for diagnosis and a target for therapy.

Spectrum of Response to Platinum and PARP Inhibitors in Germline BRCA-Associated Pancreatic Cancer in the Clinical and Preclinical Setting.

Germline BRCA-associated pancreatic ductal adenocarcinoma (glBRCA PDAC) tumors are susceptible to platinum and PARP inhibition. The clinical outcomes of 125 patients with glBRCA PDAC were stratified based on the spectrum of response to platinum/PARP inhibition: (i) refractory [overall survival (OS) <6 months], (ii) durable response followed by acquired resistance (OS <36 months), and (iii) long-term responders (OS >36 months). Patient-derived xenografts (PDX) were generated from 25 patients with glBRCA PDAC at different clinical time points. Response to platinum/PARP inhibition in vivo and ex vivo culture (EVOC) correlated with clinical response. We deciphered the mechanisms of resistance in glBRCA PDAC and identified homologous recombination (HR) proficiency and secondary mutations restoring partial functionality as the most dominant resistant mechanism. Yet, a subset of HR-deficient (HRD) patients demonstrated clinical resistance. Their tumors displayed basal-like molecular subtype and were more aneuploid. Tumor mutational burden was high in HRD PDAC and significantly higher in tumors with secondary mutations. Anti-PD-1 attenuated tumor growth in a novel humanized glBRCA PDAC PDX model. This work demonstrates the utility of preclinical models, including EVOC, to predict the response of glBRCA PDAC to treatment, which has the potential to inform time-sensitive medical decisions. SIGNIFICANCE: glBRCA PDAC has a favorable response to platinum/PARP inhibition. However, most patients develop resistance. Additional treatment options for this unique subpopulation are needed. We generated model systems in PDXs and an ex vivo system (EVOC) that faithfully recapitulate these specific clinical scenarios as a platform to investigate the mechanisms of resistance for further drug development. This article is highlighted in the In This Issue feature, p. 1749.

Pan-cancer analyses reveal cancer-type-specific fungal ecologies and bacteriome interactions.

Cancer-microbe associations have been explored for centuries, but cancer-associated fungi have rarely been examined. Here, we comprehensively characterize the cancer mycobiome within 17,401 patient tissue, blood, and plasma samples across 35 cancer types in four independent cohorts. We report fungal DNA and cells at low abundances across many major human cancers, with differences in community compositions that differ among cancer types, even when accounting for technical background. Fungal histological staining of tissue microarrays supported intratumoral presence and frequent spatial association with cancer cells and macrophages. Comparing intratumoral fungal communities with matched bacteriomes and immunomes revealed co-occurring bi-domain ecologies, often with permissive, rather than competitive, microenvironments and distinct immune responses. Clinically focused assessments suggested prognostic and diagnostic capacities of the tissue and plasma mycobiomes, even in stage I cancers, and synergistic predictive performance with bacteriomes.

Downregulation of the Tumor Suppressor TFF1 Is Required during Induction of Colon Cancer Progression by L1.

The immunoglobulin family cell adhesion receptor L1 is induced in CRC cells at the invasive front of the tumor tissue, and confers enhanced proliferation, motility, tumorigenesis, and liver metastasis. To identify putative tumor suppressors whose expression is downregulated in L1-expressing CRC cells, we blocked the L1-ezrin-NF-κB signaling pathway and searched for genes induced under these conditions. We found that TFF1, a protein involved in protecting the mucus epithelial layer of the colon, is downregulated in L1-expressing cells and displays characteristics of a tumor suppressor. Overexpression of TFF1 in L1-transfected human CRC cells blocks the pro-tumorigenic and metastatic properties conferred by L1 by suppressing NF-κB signaling. Immunohistochemical analyses revealed that human CRC tissue samples often lose the expression of TFF1, while the normal mucosa displays TFF1 in goblet cells. Identifying TFF1 as a tumor suppressor in CRC cells could provide a novel marker for L1-mediated CRC development and a potential target for therapy.

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

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.

The Collagen-Modifying Enzyme PLOD2 Is Induced and Required during L1-Mediated Colon Cancer Progression.

The overactivation of Wnt/ß-catenin signaling is a hallmark of colorectal cancer (CRC) development. We identified the cell adhesion molecule L1CAM (L1) as a target of ß-catenin-TCF transactivation in CRC cells. The overexpression of L1 in CRC cells confers enhanced proliferation, motility, tumorigenesis and liver metastasis, and L1 is exclusively localized in the invasive areas of human CRC tissue. A number of genes are induced after L1 transfection into CRC cells by a mechanism involving the cytoskeletal protein ezrin and the NF-κB pathway. When studying the changes in gene expression in CRC cells overexpressing L1 in which ezrin levels were suppressed by shRNA to ezrin, we discovered the collagen-modifying enzyme lysyl hydroxylase 2 (PLOD2) among these genes. We found that increased PLOD2 expression was required for the cellular processes conferred by L1, including enhanced proliferation, motility, tumorigenesis and liver metastasis, since the suppression of endogenous PLOD2 expression, or its enzymatic activity, blocked the enhanced tumorigenic properties conferred by L1. The mechanism involved in increased PLOD2 expression by L1 involves ezrin signaling and PLOD2 that affect the SMAD2/3 pathway. We found that PLOD2 is localized in the colonic crypts in the stem cell compartment of the normal mucosa and is found at increased levels in invasive areas of the tumor and, in some cases, throughout the tumor tissue. The therapeutic strategies to target PLOD2 expression might provide a useful approach for CRC treatment.

IRS1 phosphorylation underlies the non-stochastic probability of cancer cells to persist during EGFR inhibition therapy.

Stochastic transition of cancer cells between drug-sensitive and drug-tolerant persister phenotypes has been proposed to play a key role in non-genetic resistance to therapy. Yet, we show here that cancer cells actually possess a highly stable inherited chance to persist (CTP) during therapy. This CTP is non-stochastic, determined pre-treatment and has a unimodal distribution ranging from 0 to almost 100%. Notably, CTP is drug specific. We found that differential serine/threonine phosphorylation of the insulin receptor substrate 1 (IRS1) protein determines the CTP of lung and of head and neck cancer cells under epidermal growth factor receptor inhibition, both in vitro and in vivo. Indeed, the first-in-class IRS1 inhibitor NT219 was highly synergistic with anti-epidermal growth factor receptor therapy across multiple in vitro and in vivo models. Elucidation of drug-specific mechanisms that determine the degree and stability of cellular CTP may establish a framework for the elimination of cancer persisters, using new rationally designed drug combinations.

A Necessary Role for Increased Biglycan Expression during L1-Mediated Colon Cancer Progression.

Aberrant activation of Wnt/ß-catenin signaling and downstream ß-catenin-TCF target genes is a hallmark of colorectal cancer (CRC) development. We identified the immunoglobulin-like cell adhesion receptor L1CAM (L1) as a target of ß-catenin-TCF transactivation in CRC cells. Overexpression of L1 in CRC cells confers enhanced proliferation, motility, tumorigenesis, and liver metastasis, and L1 is exclusively localized at invasive areas of human CRC tissue. Several genes are induced after L1 transfection into CRC cells by a mechanism involving the L1-ezrin-NF-κB pathway. We conducted a secretomic analysis of the proteins in the culture medium of L1-overexpressing CRC cells. We detected a highly increased level of biglycan, a small leucine-rich ECM component, and a signaling molecule. We found that induction of biglycan is required for the cellular processes conferred by L1, including enhanced proliferation, motility, tumorigenesis, and liver metastasis. The suppression of endogenous biglycan levels or a point mutation in the L1 ectodomain that regulates cell-cell adhesion mediated by L1 blocked the enhanced tumorigenic properties conferred by L1. The mechanism of biglycan induction by L1 involves the L1-NF-κB pathway. Blocking NF-κB signaling in L1 expressing cells suppressed the induction of biglycan and the tumorigenic properties conferred by L1. Biglycan expression was undetectable in the normal colonic mucosa, but expressed at highly increased levels in the tumor tissue, especially in the stroma. The therapeutic strategies to target biglycan expression might provide a useful approach for CRC treatment in L1-overexpressing tumors.

Predicting and affecting response to cancer therapy based on pathway-level biomarkers.

Identifying robust, patient-specific, and predictive biomarkers presents a major obstacle in precision oncology. To optimize patient-specific therapeutic strategies, here we couple pathway knowledge with large-scale drug sensitivity, RNAi, and CRISPR-Cas9 screening data from 460 cell lines. Pathway activity levels are found to be strong predictive biomarkers for the essentiality of 15 proteins, including the essentiality of MAD2L1 in breast cancer patients with high BRCA-pathway activity. We also find strong predictive biomarkers for the sensitivity to 31 compounds, including BCL2 and microtubule inhibitors (MTIs). Lastly, we show that Bcl-xL inhibition can modulate the activity of a predictive biomarker pathway and re-sensitize lung cancer cells and tumors to MTI therapy. Overall, our results support the use of pathways in helping to achieve the goal of precision medicine by uncovering dozens of predictive biomarkers.

Recent insights into the role of L1CAM in cancer initiation and progression.

First described as a neuronal cell adhesion molecule, L1CAM was later identified to be present at increased levels in primary tumors and metastases of various types of cancer. Here, we describe the multifaceted roles of L1CAM that are involved in diverse fundamental steps during tumor initiation and progression, as well as in chemoresistance. Recently, Ganesh et al reported that L1CAM identifies metastasis-initiating cells in colorectal carcinoma exhibiting stem-like cell features, increased tumorigenic potential and enhanced chemoresistance. In this review, we highlight recent advances in L1CAM research with particular emphasis on its role in de-differentiation processes and cancer cell stemness supporting the view that L1CAM is a powerful prognostic factor and a suitable target for improved therapy of metastatic and drug-resistant tumors.

The human tumor microbiome is composed of tumor type-specific intracellular bacteria.

Bacteria were first detected in human tumors more than 100 years ago, but the characterization of the tumor microbiome has remained challenging because of its low biomass. We undertook a comprehensive analysis of the tumor microbiome, studying 1526 tumors and their adjacent normal tissues across seven cancer types, including breast, lung, ovary, pancreas, melanoma, bone, and brain tumors. We found that each tumor type has a distinct microbiome composition and that breast cancer has a particularly rich and diverse microbiome. The intratumor bacteria are mostly intracellular and are present in both cancer and immune cells. We also noted correlations between intratumor bacteria or their predicted functions with tumor types and subtypes, patients' smoking status, and the response to immunotherapy.

Increased expression of cathepsin D is required for L1-mediated colon cancer progression.

Hyperactivation of Wnt/ß-catenin target genes is considered a key step in human colorectal cancer (CRC) development. We previously identified the immunoglobulin-like cell adhesion receptor L1 as a target gene of ß-catenin/TCF transactivation that is localized at the invasive edge of CRC tissue. Using gene arrays, we discovered a number of downstream target genes and signaling pathways conferred by L1 overexpression during colon cancer progression. Here, we have used a proteomic approach to identify proteins in the secretome of L1-overexpressing CRC cells and studied the role of the increase in the aspartate protease cathepsin D (CTSD) in L1-mediated colon cancer development. We found that in addition to the increase in CTSD in the secretome, the RNA and protein levels of CTSD were also induced by L1 in CRC cells. CTSD overexpression resulted in elevated proliferation under stress and increased motility, tumorigenesis and liver metastasis, although to a lesser extent than after L1-transfection. The suppression of endogenous CTSD in L1-expressing cells blocked the increase in the proliferative, motile, tumorigenic and metastatic ability of CRC cells. Enhancing Wnt/ß-catenin signaling by the inhibition of GSK3ß resulted in increased endogenous CTSD levels, suggesting the involvement of the Wnt/ß-catenin pathway in CTSD expression. In human CRC tissue, CTSD was detected in epithelial cells and in the stromal compartment at the more invasive areas of the tumor, but not in the normal mucosa, indicating that CTSD plays an essential role in CRC progression.

ISG15 induction is required during L1-mediated colon cancer progression and metastasis.

Hyperactivation of Wnt/ß-catenin target gene expression is a hallmark of colorectal cancer (CRC) development. We identified L1-CAM (L1) and Nr-CAM, members of the immunoglobulin family of nerve cell adhesion receptors, as target genes of the Wnt/ß-catenin pathway in CRC cells. L1 overexpression in CRC cells enhances their motile and tumorigenic capacity and promotes liver metastasis. L1 is often localized at the invasive edge of CRC tissue. Using gene arrays and proteomic analyses we identified downstream signaling pathways and targets of L1-mediated signaling. Here, we found that the expression of interferon-stimulated gene 15 (ISG15) that operates much like ubiquitin (is conjugated to proteins by ISGylation), is elevated in the conditioned medium and in CRC cells overexpressing L1. Suppression of endogenous ISG15 levels in L1-expressing cells blocked the increased proliferative, motile, tumorigenic and liver metastatic capacities of CRC cells. ISG15 overexpression, on its own, could enhance these properties in CRC cells, but only to a much lower extent compared to L1. We show that NF-κB signaling is involved in the L1-mediated increase in ISG15, since blocking the NF-κB pathway abolished the induction of ISG15 by L1. Point mutations in the L1 ectodomain that interfere with its binding to L1 ligands, also inhibited the increase in ISG15. We detected high levels of ISG15 in human CRC tissue cells and in the adjacent stroma, but not in the normal mucosa. The results suggest that ISG15 is involved in L1-mediated CRC development and is a potential target for CRC therapy.

The intestinal stem cell regulating gene ASCL2 is required for L1-mediated colon cancer progression.

Aberrant Wnt/ß-catenin signaling is a common event during human colorectal cancer (CRC) development. Previously, we characterized members of the L1 family of cell adhesion receptors as targets of ß-catenin-LEF1/TCF transactivation that are expressed at the invasive CRC tissue edge. Overexpression of L1 in CRC cells confers enhanced motility, tumorigenesis and liver metastasis. We identified several downstream targets of L1-mediated signaling that are considered key intestinal stem cell signature genes. Here, we investigated the involvement of ASCL2, a Wnt target gene and key determinant of intestinal stem cell state, in L1-mediated CRC progression. In L1 overexpressing CRC cells we found an increase in ASCL2, a decrease in E-cadherin and accumulation of nuclear ß-catenin, ß-catenin-LEF1/TCF transactivation and target gene expression. The increase in ASCL2 by L1 overexpression enhanced ASCL2 target gene expression, conferred increased motility, tumorigenesis and metastasis, similar to L1 overexpression. Suppression of ASCL2 in cells expressing L1 blocked these tumorigenic properties. In human CRC tissue, ASCL2 was detected in the nuclei of cells at invasive areas of the tumor that also expressed L1. The results suggest that increased ASCL2 expression is a critical step in L1-mediated CRC progression.

Ex-vivo organ culture as potential prioritization tool for breast cancer targeted therapy.

The growing use of genomic testing presents new treatment options but also new dilemmas. We describe here a heavily-pretreated metastatic triple negative breast cancer patient who failed to respond to conventional treatment. Genomic analyses were performed that discovered several targetable alterations (e.g. FGFR1, CDK6, INSR) and created a clinical challenge - which target to target first? Our solution to this relatively common scenario was using ex-vivo organ culture (EVOC) system to prioritize treatment directed toward the best molecular target. EVOC enabled the trial of several potent targeted agents (Everolimus, Linsitinib, Palbociclib, AZD4547) and allowed semi-quantitative measurement of tumor response. The best response was to FGFR inhibitor, AZD4547. Consequently, the most accessible FGFR inhibiting agents (Pazopanib, then Nintedanib) were administered and some response was achieved. This report provides a potential rationale for utilizing EVOC system to predict tumor response to targeted therapy when multiple targets are proposed.

Potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine.

Growing evidence suggests that microbes can influence the efficacy of cancer therapies. By studying colon cancer models, we found that bacteria can metabolize the chemotherapeutic drug gemcitabine (2',2'-difluorodeoxycytidine) into its inactive form, 2',2'-difluorodeoxyuridine. Metabolism was dependent on the expression of a long isoform of the bacterial enzyme cytidine deaminase (CDDL), seen primarily in Gammaproteobacteria. In a colon cancer mouse model, gemcitabine resistance was induced by intratumor Gammaproteobacteria, dependent on bacterial CDDL expression, and abrogated by cotreatment with the antibiotic ciprofloxacin. Gemcitabine is commonly used to treat pancreatic ductal adenocarcinoma (PDAC), and we hypothesized that intratumor bacteria might contribute to drug resistance of these tumors. Consistent with this possibility, we found that of the 113 human PDACs that were tested, 86 (76%) were positive for bacteria, mainly Gammaproteobacteria.

The Wnt Target Gene L1 in Colon Cancer Invasion and Metastasis.

The Wnt-ß-catenin signaling pathway is highly conserved during evolution and determines normal tissue homeostasis. Hyperactivation of Wnt-ß-catenin signaling is a characteristic feature of colorectal cancer (CRC) development. ß-catenin is a major transducer of the Wnt signal from the cytoplasm into the nucleus where it acts as a co-transcriptional activator of ß-catenin-TCF target genes. ß-catenin is also required for linking cadherin type cell-cell adhesion receptors to the cytoskeleton, and consequently Wnt-ß-catenin signaling is an attractive system for investigating the role of adhesion-mediated signaling in both normal intestinal tissue homeostasis and CRC development. In this review, we summarize our studies on one Wnt-ß-catenin target gene, L1, a member of the immunoglobulin-like cell adhesion transmembrane receptor family. We describe the mechanisms of L1-mediated signaling in CRC cells, its exclusive localization in invasive areas of CRC tissue, and its ability to increase cell motility and confer metastasis to the liver. We discuss the activation (by L1) of genes via an ezrin-NF-κB pathway and the induction of genes also found in the intestinal stem cell signature. By studying L1 (adhesion)-mediated signaling, we expect to learn about mechanisms regulating both normal intestinal homeostasis and CRC development.

Clusterin, a gene enriched in intestinal stem cells, is required for L1-mediated colon cancer metastasis.

Hyperactive Wnt signaling is a common feature in human colorectal cancer (CRC) cells. A central question is the identification and role of Wnt/ß-catenin target genes in CRC and their relationship to genes enriched in colonic stem cells, since Lgr5+ intestinal stem cells were suggested to be the cell of CRC origin. Previously, we identified the neural immunoglobulin-like adhesion receptor L1 as a Wnt/ß-catenin target gene localized in cells at the invasive front of CRC tissue and showed that L1 expression in CRC cells confers enhanced motility and liver metastasis. Here, we identified the clusterin (CLU) gene that is also enriched in Lgr5+ intestinal stem cells, as a gene induced during L1-mediated CRC metastasis. The increase in CLU levels by L1 in CRC cells resulted from transactivation of CLU by STAT-1. CLU overexpression in CRC cells enhanced their motility and the reduction in CLU levels in L1 overexpressing cells suppressed the ability of L1 to confer increased tumorigenesis and liver metastasis. Genes induced during L1-mediated CRC cell metastasis and enriched in intestinal stem cells might be important for both CRC progression and colonic epithelium homeostasis.