Long-term platinum-based drug accumulation in cancer-associated fibroblasts promotes colorectal cancer progression and resistance to therapy

Cancer microenvironment; Chemotherapy; Predictive markersMicroambient del càncer; Quimioteràpia; Marcadors predictiusMicroambiente del cáncer; Quimioterapia; Marcadores predictivosA substantial proportion of cancer patients do not benefit from platinum-based chemotherapy (CT) due to the emergence of drug resistance. Here, we apply elemental imaging to the mapping of CT biodistribution after therapy in residual colorectal cancer and achieve a comprehensive analysis of the genetic program induced by oxaliplatin-based CT in the tumor microenvironment. We show that oxaliplatin is largely retained by cancer-associated fibroblasts (CAFs) long time after the treatment ceased. We determine that CT accumulation in CAFs intensifies TGF-beta activity, leading to the production of multiple factors enhancing cancer aggressiveness. We establish periostin as a stromal marker of chemotherapeutic activity intrinsically upregulated in consensus molecular subtype 4 (CMS4) tumors and highly expressed before and/or after treatment in patients unresponsive to therapy. Collectively, our study underscores the ability of CT-retaining CAFs to support cancer progression and resistance to treatment.This work has been supported by grants from Fundación científica AECC -Asociación Española contra el Cáncer- (GCAEC20030CERV) to A.Ce., from Instituto de Salud Carlos III (ISCIII) co-funded by the European Union (CP16/00151, PI17/00211, PI20/00011; Spanish Ministry of Economy and Competitiveness) to A.Ca. and PI20/00625 to P.N., from la Caixa Foundation (LCF/PR/HR19/52160018) and MICINN (PID2020-119917RB-I00) to E.B., from Spanish Ministerio de Economia y Competitividad (MINECO) and FEDER funds (PID2019-104948RB-I00) to R.R.G. This work was supported by Grant PT20/00023, funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union, and the Xarxa de Bancs de tumors sponsored by Pla Director d’Oncologia de Catalunya (XBTC). A.Ca. is the recipient of funding from the Instituto de Salud Carlos III co-funded by the European Union (MS16/00151; CPII21/00012). J.L. is the recipient of a Junior Clinician fellowship from Fundación científica AECC (CLJUN19004LINA)

Long-term platinum-based drug accumulation in cancer-associated fibroblasts promotes colorectal cancer progression and resistance to therapy

A substantial proportion of cancer patients do not benefit from platinum-based chemotherapy (CT) due to the emergence of drug resistance. Here, we apply elemental imaging to the mapping of CT biodistribution after therapy in residual colorectal cancer and achieve a comprehensive analysis of the genetic program induced by oxaliplatin-based CT in the tumor microenvironment. We show that oxaliplatin is largely retained by cancer-associated fibroblasts (CAFs) long time after the treatment ceased. We determine that CT accumulation in CAFs intensifies TGF-beta activity, leading to the production of multiple factors enhancing cancer aggressiveness. We establish periostin as a stromal marker of chemotherapeutic activity intrinsically upregulated in consensus molecular subtype 4 (CMS4) tumors and highly expressed before and/or after treatment in patients unresponsive to therapy. Collectively, our study underscores the ability of CT-retaining CAFs to support cancer progression and resistance to treatment.This work has been supported by grants from Fundación científica AECC -Asociación Española contra el Cáncer- (GCAEC20030CERV) to A.Ce., from Instituto de Salud Carlos III (ISCIII) co-funded by the European Union (CP16/00151, PI17/00211, PI20/00011; Spanish Ministry of Economy and Competitiveness) to A.Ca. and PI20/00625 to P.N., from la Caixa Foundation (LCF/PR/HR19/52160018) and MICINN (PID2020- 119917RB-I00) to E.B., from Spanish Ministerio de Economia y Competitividad (MINECO) and FEDER funds (PID2019-104948RB-I00) to R.R.G. This work was supported by Grant PT20/00023, funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union, and the Xarxa de Bancs de tumors sponsored by Pla Director d’Oncologia de Catalunya (XBTC). A.Ca. is the recipient of funding from the Instituto de Salud Carlos III co-funded by the European Union (MS16/00151; CPII21/00012). J.L. is the recipient of a Junior Clinician fellowship from Fundación científica AECC (CLJUN19004LINA)

Targeting the TGF-beta pathway of cancer-associated fibroblasts in colorectal cancer metastasis

[eng] Colorectal cancer (CRC) is the second-highest cause for cancer-related mortality worldwide. Patients suffering from CRC generally don’t die from primary tumours but rather from metastasis, for which there are no effective therapies to date. CRC progression has been correlated with the accumulation of mutations in four key signalling pathways: Wnt, MAPK, p53 and TGF-β. However, there are no relevant driver mutations described for CRC metastasis, which is produced primarily in the liver. Instead, main features of the tumour microenvironment (TME), such as T cell infiltration and overall levels of TGF-β, have acquired a prognostic value in CRC patients and can predict metastatic potential. Over the years, a better understanding on the TME has led to designing novel therapies for patients with overt metastatic disease. In the past decade, immunotherapies have represented a revolution in clinical oncology. Of note, antibodies targeting the PD-1/PD-L1 inhibitory pathway have yielded promising results in solid tumours such as melanoma, non-small-cell lung cancer and bladder cancer. Nevertheless, these therapies have failed for the majority of CRC patients, who have immunologically “cold” tumours devoid of cytotoxic T cells. In the first chapter of the present thesis, we have used a novel mouse model for metastatic CRC to describe that T cell exclusion in CRC is driven by TGF-β. By blocking the TGF-β pathway using Galunisertib, an inhibitor of the TGF-β receptor 1, activated T cells were able to infiltrate liver metastasis. Consequently, by combining treatment with Galunisertib and monoclonal antibodies against PD-L1, we were able to cure full-blown liver metastases by unleashing a potent T cell-mediated cytotoxic response. Despite the results obtained in chapter 1, the specific cellular mechanisms of this TGF- β-mediated T cell exclusion needed further elucidation. In this regard, our lab has reported that TGF-β leads to the expression of a gene signature in cancer-associated fibroblasts (CAFs) that predicts relapse in patients, and that CAFs are crucial for CRC tumour survival and metastatic colonisation. Therefore, we asked whether TGF-β- activated CAFs were also responsible for T cell exclusion in liver metastases. The work conducted to tackle this question is divided in two chapters. In chapter 2, we have focused on investigating the biology of CAFs from CRC liver metastases, resulting in the establishment of specific markers to target fibroblasts. Moreover, we have defined two different CAF subpopulations coexisting in CRC liver metastases, one of which could be directly related with T cell exclusion. In chapter 3, we have established a genetic mouse model of CRE-LoxP-mediated recombination to specifically ablate the TGF-β receptor 2 in CAFs from liver metastases. CRE expression was driven by the promoter of Transgelin, a TGF-β target gene expressed in CAFs that strongly correlates with relapse in CRC patients. Abrogation of the TGF-β pathway in CAFs did not alter T cell infiltration in metastases. Nevertheless, combination of genetic ablation of the TGF-β receptor 2 and treatment with blocking antibodies against PD-L1 led to curative responses, strongly suggesting that TGF-β- activated CAFs are crucial for mediating T cell exclusion in metastases and evading checkpoint immunotherapy.[cat] El càncer colorrectal (CCR) és el segon càncer més mortal a nivell mundial. Els pacients amb CCR generalment no moren degut als tumors primaris, sinó degut a la metàstasi, per la qual no existeix cap tractament efectiu. La progressió del CCR està correlacionada a la acumulació de mutacions en quatre rutes de senyalització clau: Wnt, MAPK, p53 i TGF-β. Tanmateix, no hi ha cap mutació rellevant descrita per a la metàstasi de CCR, la qual es produeix principalment al fetge. En canvi, determinats trets del microambient tumoral (MAT), com ara la infiltració de cèl·lules T i els nivells globals de TGF-β, han adquirit un valor important en el pronòstic de pacients amb CCR i poden predir el risc de metàstasi. Al llarg dels anys, entendre amb profunditat el MAT ha portat a dissenyar noves teràpies per a pacients amb metàstasis. Durant la darrera dècada, les immunoteràpies han representat una revolució per a la oncologia clínica. En particular, els anticossos que bloquegen la ruta inhibitòria PD-1/PD-L1 han aportat resultats prometedors en pacients amb tumors sòlids com ara melanoma, càncer de pulmó i de bufeta. Tanmateix, aquestes teràpies han fallat per a la majoria de pacients amb CCR, els quals generen tumors “freds”, amb manca de cèl·lules T. En el primer capítol de la tesi present, hem emprat un nou model murí de CCR metastàtic per a descriure que l’exclusió de cèl·lules T és deguda a TGF-β. El bloqueig de la ruta de TGF-β emprant Galunisertib, un inhibidor del receptor 1 de TGF-β, permet la infiltració de cèl·lules T activades dins de les metàstasis del fetge. Posteriorment, quan es combina l’acció de Galunisertib amb anticossos monoclonals contra PD-L1, vam ser capaços de promoure una resposta citotòxica potent capaç de curar metàstasis hepàtiques. Malgrat els resultats presentats en el capítol 1, encara necessitàvem conèixer els mecanismes cel·lulars específics d’aquesta exclusió de cèl·lules T controlada per TGF- β. En aquest aspecte, el nostre laboratori va demostrar que TGF-β permet l’expressió d’una signatura genètica expressada en fibroblasts associats a cèncer (FACs) que prediuen remissions a pacients. A més, va demostrar que els FACs són crucials per a la supervivència dels tumors i la colonització a altres òrgans. Per tant, ens vam preguntar si els FACs activats per TGF-β també son responsables de la exclusió de cèl·lules T en metàstasis hepàtiques. La feina per adreçar aquesta pregunta es divideix en dos capítols. En el capítol 2, ens vam centrar en investigar la biologia dels FACs de les metàstasis, i hem establert marcadors específics per estudiar els fibroblasts. A més, hem definit dues subpoblacions de FACs que coexisteixen en les metàstasis. Possiblement, una d’aquestes subpoblacions és reponsable de la exclusió de cèl·lules T. En el capítol 3, hem establert un model murí de recombinació per CRE-LoxP del receptor 2 de TGF-β en FACs. L’expressió de la CRE estava controlada pel promotor de Transgelin, un gen diana de TGF-β expressat en FACs i que està molt relacionat amb remissions de CCR a pacients. L’eliminació de la ruta del TGF-β a FACs no va alterar la infiltració de les cèl·lules T. Tot i així, la combinació de la eliminació genètica del receptor 2 de TGF-β amb anticossos contra PD-L1 va proporcionar respostes curatives, la qual cosa suggereix que els FACs activats per TGF-β són realment crucials per controlar l’exclusió de cèl·lules T en metàstasis hepàtiques i per la resistència a immunoteràpia

Minimal Residual Disease, Metastasis and Immunity

Progression from localized to metastatic disease requires cancer cells spreading to distant organs through the bloodstream. Only a small proportion of these circulating tumor cells (CTCs) survives dissemination due to anoikis, shear forces and elimination by the immune system. However, all metastases originate from CTCs capable of surviving and extravasating into distant tissue to re-initiate a tumor. Metastasis initiation is not always immediate as disseminated tumor cells (DTCs) may enter a non-dividing state of cell dormancy. Cancer dormancy is a reversible condition that can be maintained for many years without being clinically detectable. Subsequently, late disease relapses are thought to be due to cancer cells ultimately escaping from dormant state. Cancer dormancy is usually associated with minimal residual disease (MRD), where DTCs persist after intended curative therapy. Thus, MRD is commonly regarded as an indicator of poor prognosis in all cancers. In this review, we examine the current understanding of MRD and immunity during cancer progression to metastasis and discuss clinical perspectives for oncology

Minimal residual disease, metastasis and immunity

Progression from localized to metastatic disease requires cancer cells spreading to distant organs through the bloodstream. Only a small proportion of these circulating tumor cells (CTCs) survives dissemination due to anoikis, shear forces and elimination by the immune system. However, all metastases originate from CTCs capable of surviving and extravasating into distant tissue to re-initiate a tumor. Metastasis initiation is not always immediate as disseminated tumor cells (DTCs) may enter a non-dividing state of cell dormancy. Cancer dormancy is a reversible condition that can be maintained for many years without being clinically detectable. Subsequently, late disease relapses are thought to be due to cancer cells ultimately escaping from dormant state. Cancer dormancy is usually associated with minimal residual disease (MRD), where DTCs persist after intended curative therapy. Thus, MRD is commonly regarded as an indicator of poor prognosis in all cancers. In this review, we examine the current understanding of MRD and immunity during cancer progression to metastasis and discuss clinical perspectives for oncology

Determinants and functions of CAFs secretome during cancer progression and therapy

Multiple lines of evidence are indicating that cancer development and malignant progression are not exclusively epithelial cancer cell-autonomous processes but may also depend on crosstalk with the surrounding tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are abundantly represented in the TME and are continuously interacting with cancer cells. CAFs are regulating key mechanisms during progression to metastasis and response to treatment by enhancing cancer cells survival and aggressiveness. The latest advances in CAFs biology are pointing to CAFs-secreted factors as druggable targets and companion tools for cancer diagnosis and prognosis. Especially, extensive research conducted in the recent years has underscored the potential of several cytokines as actionable biomarkers that are currently evaluated in the clinical setting. In this review, we explore the current understanding of CAFs secretome determinants and functions to discuss their clinical implication in oncology

Challenges and Therapeutic Opportunities in the dMMR/MSI-H Colorectal Cancer Landscape

About 5 to 15% of all colorectal cancers harbor mismatch repair deficient/microsatellite instability–high status (dMMR/MSI-H) that associates with high tumor mutation burden and increased immunogenicity. As a result, and in contrast to other colorectal cancer phenotypes, a significant subset of dMMR/MSI-H cancer patients strongly benefit from immunotherapy. Yet, a large proportion of these tumors remain unresponsive to any immuno-modulating treatment. For this reason, current efforts are focused on the characterization of resistance mechanisms and the identification of predictive biomarkers to guide therapeutic decision-making. Here, we provide an overview on the new advances related to the diagnosis and definition of dMMR/MSI-H status and focus on the distinct clinical, functional, and molecular cues that associate with dMMR/MSI-H colorectal cancer. We review the development of novel predictive factors of response or resistance to immunotherapy and their potential application in the clinical setting. Finally, we discuss current and emerging strategies applied to the treatment of localized and metastatic dMMR/MSI-H colorectal tumors in the neoadjuvant and adjuvant setting

The prognostic potential of CDX2 in colorectal cancer: Harmonizing biology and clinical practice

Adjuvant chemotherapy following surgical intervention remains the primary treatment option for patients with localized colorectal cancer (CRC). However, a significant proportion of patients will have an unfavorable outcome after current forms of chemotherapy. While reflecting the increasing complexity of CRC, the clinical application of molecular biomarkers provides information that can be utilized to guide therapeutic strategies. Among these, caudal-related homeobox transcription factor 2 (CDX2) emerges as a biomarker of both prognosis and relapse after therapy. CDX2 is a key transcription factor that controls intestinal fate. Although rarely mutated in CRC, loss of CDX2 expression has been reported mostly in right-sided, microsatellite-unstable tumors and is associated with aggressive carcinomas. The pathological assessment of CDX2 by immunohistochemistry can thus identify patients with high-risk CRC, but the evaluation of CDX2 expression remains challenging in a substantial proportion of patients. In this review, we discuss the roles of CDX2 in homeostasis and CRC and the alterations that lead to protein expression loss. Furthermore, we review the clinical significance of CDX2 assessment, with a particular focus on its current use as a biomarker for pathological evaluation and clinical decision-making. Finally, we attempt to clarify the molecular implications of CDX2 deficiency, ultimately providing insights for a more precise evaluation of CDX2 protein expression

Oikopleura dioica culturing made easy:a low-cost facility for an emerging animal model in EvoDevo

The genome sequencing and the development of RNAi knockdown technologies in the urochordate Oikopleura dioica are making this organism an attractive emergent model in the field of EvoDevo. To succeed as a new animal model, however, an organism needs to be easily and affordably cultured in the laboratory. Nowadays, there are only two facilities in the world capable to indefinitely maintain Oikopleura dioica, one in the SARS institute (Bergen, Norway) and the other in the Osaka University (Japan). Here, we describe the setup of a new facility in the University of Barcelona (Spain) in which we have modified previously published husbandry protocols to optimize the weekly production of thousands of embryos and hundreds of mature animals using the minimum amount of space, human resources, and technical equipment. This optimization includes novel protocols of cryopreservation and solid cultures for long-term maintenance of microalgal stocks-Chaetoceros calcitrans, Isochrysis sp., Rhinomonas reticulate, and Synechococcus sp.-needed for Oikopleura dioica feeding. Our culture system maintains partially inbred lines healthy with similar characteristics to wild animals, and it is easily expandable to satisfy on demand the needs of any laboratory that may wish to use Oikopleura dioica as a model organism.</p

TGFβ drives immune evasion in genetically reconstituted colon cancer metastasis

Most patients with colorectal cancer die as a result of the disease spreading to other organs. However, no prevalent mutations have been associated with metastatic colorectal cancers1,2. Instead, particular features of the tumour microenvironment, such as lack of T-cell infiltration3, low type 1 T-helper cell (TH1) activity and reduced immune cytotoxicity2 or increased TGFβ levels4 predict adverse outcomes in patients with colorectal cancer. Here we analyse the interplay between genetic alterations and the tumour microenvironment by crossing mice bearing conditional alleles of four main colorectal cancer mutations in intestinal stem cells. Quadruple-mutant mice developed metastatic intestinal tumours that display key hallmarks of human microsatellite-stable colorectal cancers, including low mutational burden5, T-cell exclusion3 and TGFβ-activated stroma4,6,7. Inhibition of the PD-1–PD-L1 immune checkpoint provoked a limited response in this model system. By contrast, inhibition of TGFβ unleashed a potent and enduring cytotoxic T-cell response against tumour cells that prevented metastasis. In mice with progressive liver metastatic disease, blockade of TGFβ signalling rendered tumours susceptible to anti-PD-1–PD-L1 therapy. Our data show that increased TGFβ in the tumour microenvironment represents a primary mechanism of immune evasion that promotes T-cell exclusion and blocks acquisition of the TH1-effector phenotype. Immunotherapies directed against TGFβ signalling may therefore have broad applications in treating patients with advanced colorectal cancer