Dr. Weeks’ Comment: Readers of WeeksMD have known for decades that chemotherapy is more harmful than beneficial – “Friends don’t let Friends get Chemo” – has been a rallying cry for those who understand the lethality not of the cancer TUMOR cell but of the cancer STEM cell. We taught that chemo and radiation therapy make your cancer worse – they make the lethal cancer STEM cells more numerous and more virulent. But now we learn that chemo itself increase the life threatening metastasis. We knew that inflammation increases metastasis but now we see chemo itself does this disservice to trusting patients. Make certain your oncologist knows this.

Neoadjuvant chemotherapy induces breast cancer metastasis through a TMEM-mediated mechanism

1. George S. Karagiannis et al

• FULL Article https://stm.sciencemag.org/content/9/397/eaan0026

Closing the door to cancer cells

Breast cancer is one of the most common tumor types, and metastasis greatly increases the risk of death from this disease. By studying the process of intravasation or entry of cells into the vasculature, Karagiannis et al. discovered that, in addition to killing tumor cells, chemotherapy treatment can also increase intravasation. Groups of cells collectively known as tumor microenvironment of metastasis (TMEM) can serve as gateways for tumor cells entering the vasculature, and the authors discovered that several types of chemotherapy can increase the amounts of TMEM complexes and circulating tumor cells in the bloodstream. The researchers also determined that a drug called rebastinib can interfere with TMEM activity and help overcome the increased risk of cancer cell dissemination.

Abstract

Breast cancer cells disseminate through TIE2/MENA^Calc/MENA^INV-dependent cancer cell intravasation sites, called tumor microenvironment of metastasis (TMEM), which are clinically validated as prognostic markers of metastasis in breast cancer patients. Using fixed tissue and intravital imaging of a PyMT murine model and patient-derived xenografts, we show that chemotherapy increases the density and activity of TMEM sites and Mena expression and promotes distant metastasis. Moreover, in the residual breast cancers of patients treated with neoadjuvant paclitaxel after doxorubicin plus cyclophosphamide, TMEM score and its mechanistically connected MENA^INV isoform expression pattern were both increased, suggesting that chemotherapy, despite decreasing tumor size, increases the risk of metastatic dissemination. Chemotherapy-induced TMEM activity and cancer cell dissemination were reversed by either administration of the TIE2 inhibitor rebastinib or knockdown of the MENAgene. Our results indicate that TMEM score increases and MENA isoform expression pattern changes with chemotherapy and can be used in predicting prometastatic changes in response to chemotherapy. Furthermore, inhibitors of TMEM function may improve clinical benefits of chemotherapy in the neoadjuvant setting or in metastatic disease.

DISCUSSION

Accumulating evidence indicates that chemotherapy evokes a host repair response, during which bone marrow–derived cells (BMDCs) infiltrate the primary tumor microenvironment and facilitate neoangiogenesis and tumor regrowth (10, 11). Here, we have shown that through such BMDC recruitment, neoadjuvant chemotherapy (NAC) may increase cancer cell dissemination and induce a more aggressive tumor phenotype with increased metastasis. The mechanism involves both the assembly of TMEM sites and the increased MENA^INV expression in residual cancer after NAC. These results are consistent with our previous findings that MENA expression is required for TMEM assembly and for cancer cell dissemination through a TMEM MENA^INV and TIE2^hi/VEGF^hi macrophage-dependent mechanism (1, 20, 21, 29). Although the effects of taxanes and other chemotherapeutics on neovascularization have been adequately described (8, 10, 12, 13, 35, 43), our study provides insight into the mechanisms by which paclitaxel and other chemotherapies modulate the cancer microenvironment to promote breast cancer cell intravasation and dissemination to distant sites, as well as a TIE2-directed therapeutic approach to counteract paclitaxel-mediated induction of cancer cell dissemination (Fig. 8K). Thus, this work is primarily focused on the chemotherapy effect on cancer cell dissemination via TMEM/MENA-mediated mechanism.

We demonstrated that chemotherapy increases macrophage density in a PDX model but not in spontaneous PyMT. Our findings in the PyMT model are in discrepancy with the Coussens group (44). This may be because we worked with 8- to 9-week-old mice bearing early-stage spontaneous carcinomas, whereas the Coussens group worked with 12-week-old mice, which typically have advanced-stage tumors. However, our findings demonstrated that chemotherapy promotes an increase in perivascular TIE2^hi/VEGF^hi macrophages (Fig. 8K), which is consistent with studies showing that this population associates with sites of (patho)physiological angiogenesis, especially as a host repair mechanism after cytotoxic damage through chemotherapy (14, 15, 45–47). Although it is not clear whether TIE2^hi/VEGF^hi macrophages belong to the “classically activated” (M1) or “alternatively activated” (M2) group, they are crucial for modulating the tumor microenvironment in response to cytotoxic therapies (10, 48). Our observation that other chemotherapeutics, such as doxorubicin/ cyclophosphamide, are capable of perivascular TIE2^hi/VEGF^hi macrophage recruitment, TMEM assembly, and TMEM-dependent tumor cell intravasation further supports the idea that the mechanism by which chemotherapy induces these prometastatic effects is a generic host repair mechanism in response to extensive tissue damage and not a paclitaxel-specific phenomenon. For instance, TIE2⁺ macrophages also express the chemokine receptor CXCR4, and chemotherapy may increase the expression of the CXCR4 ligand CXCL12 in the primary tumor microenvironment (10). Therefore, it is very likely that the prometastatic TIE2^hi/VEGF^hi macrophages are recruited through a distinct chemotactic axis in chemotherapy-treated individuals.

In addition, increased macrophage infiltration into tumors upon paclitaxel treatment increases the contact between tumor cells and macrophages, which is known to stimulate the expression of MENA^INV via NOTCH pathway activation, resulting in increased MENA^INV and TMEM-dependent intravasation (27). These observations suggest that paclitaxel treatment may have induced MENA^INV and MENA^Calc expression due to chemotherapy-driven macrophage infiltration, resulting in increased TMEM assembly and function as described here (Fig. 8K). This may be an active process and not simply the result of selective survival of MENA-expressing tumor cells during paclitaxel treatment (49). Otherwise, our data are in agreement with findings from Oudin et al. (49), who showed increased MENA and MENA^INV expression in paclitaxel-treated compared to control MDA-MB-231 xenografts.

The observed increase in disseminating tumor cells upon chemotherapy treatment as a direct consequence of macrophage contact–induced MENA^INV overexpression is supported by two key findings reported here. First, MENA^INV and MENA^Calc expression correlated especially well with TIE2^hi macrophages, consistent with earlier studies in humans and in mice (18, 20, 29). Second, the absence of all MENA isoforms completely abolished cancer cell dissemination and distant metastasis in vivo, regardless of whether those mice received paclitaxel or not and without affecting TIE2^hi/VEGF^hi macrophage recruitment, indicating that MENA expression is an essential prerequisite for paclitaxel-induced breast cancer cell transendothelial migration in vivo.

Our study indicates that the TMEM score and MENA^INV increase in breast cancer samples from patients treated with NAC including doxorubicin, cyclophosphamide, and paclitaxel, suggesting that TMEM score and MENA^INV might be used in predicting development of prometastatic changes in primary tumor microenvironment in response to NAC. This is important because many breast cancer patients are treated with NAC, which typically lasts about 6 months, and currently, there are no markers that predict response to NAC (43). Our data indicate that in patients who have RCB post-NAC, such as those with ER⁺ disease, NAC could induce metastases via TMEM, despite inducing partial tumor regression. In particular, only 16.5% of patients with ER⁺/HER2⁻disease achieve pathologic complete response (pCR) with NAC, indicating that our findings may apply to the majority of patients, that is, those who do not achieve pCR (50). Although addition of paclitaxel to NAC increases the percentage of patients with pCR, it does not improve the overall survival (6, 7), suggesting that some patients do not draw long-term benefit from NAC. Because we showed that after only two doses of chemotherapy, MENA^INV increases in some patients, we speculate that MENA isoform expression status in FNA biopsy after the first 2 weeks of chemotherapy could predict which patients would receive full benefit from NAC and in which continuation of NAC would be harmful. For example, an approach could be developed to routinely assess the expression of MENA^INV in FNA samples after the second chemotherapeutic dose. If the invasive isoforms of MENA do not increase, then the chemotherapy could be continued to its completion. Conversely, if there is an increase in the invasive MENA isoform, then the chemotherapy could be discontinued and these patients could be treated with surgery first, followed by chemotherapy. However, the effectiveness of such an approach would need to be investigated in future studies…

Our cohort of 20 patients with ER⁺ disease had only 5 years of follow-up time, which is not sufficient to reliably analyze distant recurrence in this breast cancer subtype because ER⁺ disease often recurs 10 or more years after the initial diagnosis (51). However, two retrospective-prospective analyses of human breast cancer samples indicate that increased TMEM score is associated with metastatic outcome in patients (3, 5). These studies imply that with the proper follow-up time, the increase in TMEM score upon chemotherapy should translate into distant recurrence in some of the patients. A follow-up study is needed to determine whether patients with an increase in TMEM score upon NAC develop distant recurrence more often than those without an increase in TMEM score. In addition, as discussed above, it is necessary to determine whether we can predict which patients are likely to respond to NAC by assessing MENA^INV so that the treatment can be adjusted accordingly.

Our finding that paclitaxel induces an increase in CTCs is consistent with recently reported data from patient studies focused on the effect of chemotherapy on CTCs. Although CTC count measured by U.S. Food and Drug Administration–approved CellSearch System is a strong prognostic factor in both primary and metastatic breast cancer, there is no conclusive evidence in the literature that chemotherapy significantly reduces CTCs (52). On the contrary, several reports indicate that CTC counts in post-chemotherapy blood samples increase in some patients and decrease in others, do not correlate with pCR, and correlate with distant metastasis–free survival (53, 54). Moreover, when CTC search included cells with epithelial-mesenchymal transition marker expression, 21% of patients showed increased CTC counts after NAC, whereas 15% showed a decrease in CTCs counts after NAC (54). Thus, our data indicating that NAC may be increasing CTCs in some patients are consistent with current literature.