Revolutionizing breast cancer recurrence prevention using ablation

Introduction: : Breast cancer care presents a ripe opportunity for disruptive innovation. Of the more than 2 million patients diagnosed with breast cancer each year, more than half of them will undergo surgery as their first line treatment. Unfortunately, up to 40% of these patients will experience recurrence in the form of metastatic disease. One reason for recurrence is a lack of immune memory to fend off future disease, primarily because the in-situ tumor was cloaked from immune detection and removed by surgery before any disruption that could have promoted lasting anti-tumor immunity. We have developed a low-cost, minimally invasive percutaneous ablation approach, called PROTECT (Polymer-based Recurrence prevention through an Optimal Targeted Ethanol Cancer Treatment) and demonstrated in triple negative breast cancer (TNBC) that treatment with PROTECT has the potential to prevent future recurrence. PROTECT consists of a polymer backbone (ethyl cellulose) dissolved in ethanol (cell kill) that forms an ethanol-rich gel upon injection into tissue. The polymer backbone creates tunability for different size injections depending on tissue mechanical properties. Here, we performed two sets of in vivo experiments in orthotopic models of TNBC to investigate the impacts of PROTECT in combination with immune modulating agents like cyclophosphamide (CP) and immune checkpoint blockade (ICB) on tumor growth, metastases, overall survival, and the anti-tumor immune response.

Materials and

Methods: : Our first experiment investigated which combination of therapies performed best in terms of overall survival and metastases. BALB/c mice were implanted with orthotopic 4T1-Luc tumors. When tumors were palpable (~day 10), mice were randomized to one of 3 treatment groups (PROTECT + CP, PROTECT + anti-PD1, PROTECT + CP + anti-PD1) or 5 control groups (see Figure 1A) with approximately 10 mice per group. Mice received treatment based on the timeline shown in Figure 1A. PROTECT treatment was performed via direct injection of 150 L 10% w/w ethyl cellulose in ethanol. CP and anti-PD1 were delivered subcutaneously at doses of 200 mg/kg and 20 mg/kg, respectively. Mice were euthanized at a humane endpoint (e.g., tumor volume of ~1500 mm3) or 60 days, whichever occurred first.
We selected a subset of treatments, including PROTECT + CP + anti-PD1 and PROTECT + CP, from our first study to investigate in a second study to mimic use clinically as a neoadjuvant therapy (Figure 1B). BALB/c mice bearing orthotopic 4T1-Luc tumors were randomized to one of two treatment groups or saline control, with n = 9 mice per group. Treatment was performed as described above. Mice underwent treatment and IVIS imaging, to monitor tumor growth and metastasis formation, per Figure 1B. The primary tumor was resected for analysis by IHC (CD4 and CD8) and flow cytometry (CD45, CD3, CD4, and CD8) ~23 days after PROTECT treatment.

Results, Conclusions, and Discussions:: We first investigated how PROTECT in combination with CP and anti-PD1 ICB impacted overall survival and metastases in 4T1-Luc TNBC (Figure 2). PROTECT + CP + anti-PD1 significantly reduced primary tumor growth compared to saline controls, translating into a significant increase in overall survival. Importantly, of the 12 mice treated with the triple therapy, three (25%) of them were cured, defined as no evidence of primary tumor or metastases at 60 days. The second-best performing therapy was PROTECT + CP, which also resulted in a significant increase in overall survival compared to saline controls. These two therapies were selected as candidates for our second experiment investigating PROTECT as a neoadjuvant therapy.
Our neoadjuvant therapy study examined how the addition of PROTECT + CP with or without ICB impacted the tumor immune microenvironment and the development of metastases. Through IVIS imaging leading up to primary tumor surgical excision, we found that mice receiving either treatment showed no evidence of lung metastases development compared to control mice (Figure 3A). Primary tumor volume and IVIS flux both demonstrated that primary tumor growth was significantly reduced in both treatment groups (Figure 3B,C).
Assessment of the primary tumor for CD3, CD4 and CD8 expression by flow cytometry (Figure 3D) and IHC (Figure 3E) showed that T-cell infiltration into the tumor was significantly higher for treatment with PROTECT + CP with and without ICB compared to saline controls. The increase in T cells conferred survival advantages following surgery both in terms of increased time to distant recurrence (Figure 3F) and overall survival (Figure 3G).
Our results demonstrate neoadjuvant treatment with PROTECT and immune modulators alters the tumor immune microenvironment in a manner consistent with lasting anti-tumor immunity. Given the results of I-SPY phase II and KEYNOTE 522 revealing an increase in pathologic complete response following neoadjuvant chemotherapy with ICB, PROTECT offers a complementary approach in the neoadjuvant setting with the added benefit of tumor ablation.

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