Abstract
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.
Introduction
More than four decades after their first approval, platinum drugs still stand among the most widely utilized anti-cancer agents1. Nowadays, almost 50% of cancer patients receiving chemotherapy (CT) are indeed treated with systemic platinum-based regimen1,2. Notwithstanding platinum drugs’ effectiveness in eradicating cancer cells by means of adducts and crosslinks accumulation in the DNA, a large number of tumors are able to bypass the cytotoxic effect of platinum through primary or acquired resistance1. In colorectal cancer (CRC), the improvement in survival provided by oxaliplatin-based regimens is estimated to be no more than 20% in stage III and less than 5% in stage II localized cancer patients3,4. Similarly, metastatic CRC patients that are initially responding to therapy often experience disease progression due to the emergence of acquired drug-resistance5.
For these reasons, elucidating the determinants of platinum-based treatment effectiveness is paramount to improve the standard of care for cancer patients. Much effort has been made to decipher the mechanisms of platinum resistance intrinsic to epithelial cancer cells1,6 and multiple molecular classifications were developed in the recent years, enabling the identification of CRC subtypes associated with worse patient’s outcome7,8,9,10 and lack of benefit from CT, including oxaliplatin10,11. Initially, there was no apparent molecular consensus between the poor prognostic subtypes of CRC, which were either enriched in Wnt signaling and markers associated with cancer stem cells (Stem-like)7, in serrated tumors (CCS3)9, or in mesenchymal tumors with deficient mismatch repair machinery (C-type)10. In an effort to integrate the previously published classification algorithms, Guinney and colleagues defined four Consensus Molecular Subtypes (CMS) of CRC, namely CMS1 (MSI immune), CMS2 (canonical), CMS3 (metabolic) and CMS4 (mesenchymal), from which CMS4 tumors showed the worse clinical outcome8. However, these classification systems and advances in the genetics of cancer have not yet translated into efficient treatment synergizing with platinum drugs and failed so far to provide biomarkers of therapeutic response12,13.
Importantly, cancer cells do not exist as isolated entities, but rather reside in an interactive tumor microenvironment (TME) composed of non-malignant cells that largely contributes to cancer progression. For instance, the activation of the TGF-beta pathway in cancer-associated fibroblasts (CAFs) populating the TME is considered as a hallmark of worse prognosis for CRC patients whereas low stromal TGF-beta activity associates with increased disease-free survival14. In this line, growing evidence indicates that systemic CT affects non-malignant cells in the TME of cancers from distinct origin15,16. However, the mechanisms through which CT regulates stromal functions and to what extent these processes influence cancer progression as well as patient’s susceptibility to treatment remain unclear.
In this work, we assess the presence of oxaliplatin in residual tumors after treatment and investigate the impact of platinum-based therapy on stroma-originating pro-oncogenic signaling. Surprisingly, we observe that significant amount of oxaliplatin is retained by the TME long time after CRC patient treatment. We functionally dissect the influence of platinum-stimulated TME on cancer progression and demonstrate that the accumulation of platinum in CAFs participates to the acquisition of stromal cues that associate with increased cancer aggressiveness and resistance to treatment.
Results
Platinum-based drug accumulates in fibroblasts resilient to treatment
We first aimed to investigate the immediate impact of CT on the TME. For this, we used a model of aggressive CRC grown from mouse tumor organoids (MTO) carrying compound genetic alterations (Apc, Kras, Trp53, Tgfbr2) injected into the caecum wall of immunocompetent C57BL/6J mice (see “Methods”)17. Mice were administered with oxaliplatin 96 and 24 h before tumor resection (see “Methods”). Bioluminescence tracking in vivo showed a reduction of cancer cells abundance upon treatment (Fig. 1a). Following resection, tumor samples were analyzed by immunohistochemistry (IHC) to assess α-SMA (+) CAFs, CD31 (+) endothelial and CD45 (+) immune cells abundance. We observed a significant reduction of blood vessels density and immune cells infiltration following therapy (Fig. 1a and Supplementary Fig. 1a). On the other hand, CAFs abundance remained unchanged (Fig. 1a and Supplementary Fig. 1a) therefore suggesting that in contrast to cancer, endothelial and immune cells, CAFs are highly resistant to oxaliplatin. We next assessed oxaliplatin cytotoxicity against cultured colonic fibroblasts (CCD-18Co), CAFs derived from CRC patients (CAF1, CAF2, CAF3), immune cells (PBMC), endothelial cells (HUVEC), CRC cell line (HT29-M6) and patient-derived CRC organoids (PDO, PDO2). In accordance with in vivo findings, we observed that CRC (Fig. 1b and Supplementary Fig. 1b), immune (Supplementary Fig. 1c) and endothelial cells (Supplementary Fig. 1d) were highly sensitive to oxaliplatin compared to fibroblasts (Fig. 1b and Supplementary Fig. 1e). In order to determine the tumor cells response to CT over time, we treated cultured CCD-18Co, CAF1 and CAF2 as well as HT29-M6 cells, PDO and PDO2 with oxaliplatin for 12 days. Cancer cells did not survive to 9 days of treatment (Fig. 1c and Supplementary Fig. 1f). In contrast, about 50–80% of fibroblasts resisted to up to 12 days CT (Fig. 1c and Supplementary Fig. 1f). Yet, ICP-MS analysis indicated an increased oxaliplatin absorption in cultured fibroblasts compared to CRC cells (Fig. 1d). Interestingly, traces of platinum were still detectable in fibroblasts long after oxaliplatin retrieval (Fig. 1d)….
