Collectively, our results demonstrate the remarkable extent to which HB1.F3.CD21 NSCs rapidly and selectively home to an overwhelming majority of peritoneal metastases. Here, we discuss our major findings in the context of current literature.
IP administered NSCs Localize Efficiently to Peritoneal Metastases: Although IP-administered NSCs have the potential to migrate anywhere on the vast peritoneal surface area (1.7 m2), our ICP-MS based quantification of NSC-associated AuNRs unexpectedly showed that an impressive 70–80% of NSCs localized to tumors, reaching up to 95% within 3 h after injection (Fig.2B,3A). One possible explanation for this tumor-specific localization is that the healthy mesothelium is protected by a layer of anti-adhesive proteoglycans, whereas tumor disruptions expose the underlying basement membrane, revealing extracellular matrix substrates (e.g., collagen I/IV, fibronectin, hyaluronan, and laminin) that can be bound by CD44 (26) and α5β1 integrins (27) expressed by the HB1.F3.CD.21 NSCs. Because this study focused only on intra-abdominal tumors and peritoneal lavage fluid, we were unable to account for the distribution of approximately 20% of injected NSCs. A minority of these NSCs may have been cleared from the abdomen through either the general circulation or lymphatic systems.
The behavior of NSCs, including their pharmacokinetic and biological properties, can be affected by the mode of administration (28–30). While intravenous administration offers several clinical advantages including ease of delivery and access to systemic metastasis, local IP administration is often pursued in the ovarian cancer setting to increasing the bioavailability of treatments at the target site. For example, a landmark clinical trial reported a significant survival benefit for ovarian cancer patients who had undergone a combination of intravenous (IV) and intraperitoneal (IP) cisplatin and paclitaxel as compared to IV paclitaxel and cisplatin alone(31). Local IP administration was chosen for the current study because most stem cell-based therapies that have progressed to late-stage clinical trials have used local administration (i.e., intracranial, intrathecal, intralesional, and endocardial)(31). Furthermore, we have previously demonstrated that that when NSCs are administered IV rather than IP, little to no NSCs are observed in tumors in the IP cavity (14). Systemic administration of stem cells still has key challenges including the instant blood-mediated inflammatory reaction (triggers coagulation), the pulmonary bypass barrier, and insufficient residence time at the target site(32).
We used the unconventional method of quantifying endocytosed AuNRs to monitor NSC tropism in order to overcome the weaknesses of existing cell-tracking technologies. Although the NSCs are engineered to express both eGFP and ffluc, given the resolution limits of live-animal imaging, these markers do not permit sensitive, quantitative NSC detection. Live animal imaging for ovarian tumor models can also be particularly troublesome as the depth and location of the tumors around the IP organs can limit the signal detection.. Furthermore, tumor and stromal DNA/cell counts overwhelm NSC-specific signals, so the number of NSCs present in tumor tissues is not reliably discernable using PCR or FACS. In addition, membrane dyes cannot be used without a complementary approach due to the possibility of dye transfer from injected cells to host cells as well as the possibility of photo-bleaching and fluorophore instability after fixative methods (33). In contrast, inorganic NP trackers provide a better balance of sensitivity, dynamic range, and stability for assessing the distribution of IP-administered NSCs as there is no background signal to hinder quantification. Importantly, this method also enables the macroscopic observation of NSC migration, as well as the ability to assess NSC cargo delivery (Fig. 1A,2A).
NSCs Provide an Efficient Therapeutic Cargo Delivery System: Our results demonstrate that the impressive tumor tropism of NSCs to IP metastases may significantly advance peritoneal chemotherapy by guiding the delivery of pre-loaded therapeutic cargo. We observed that, whereas the localization of NPs to tumors is low when they are delivered freely (perhaps because they are engulfed by peritoneal or tumor-associated macrophages), their tumor localization was significantly (more than 60%) greater when they were delivered within NSCs (Fig. 1D). We believe that this improvement can be generalized to other free vs. NSC-delivered therapeutic payloads. For example, we have previously reported improved NSC-mediated drug delivery of two standard-of-care chemotherapeutic drugs, cisplatin (14) and paclitaxel (PTX)(15) to peritoneal metastases; as well as two oncolytic viruses (34,35). This delivery system can not only improve tumor localization, but also potentially reduce toxicity and prolong release of therapeutic reagent.
One critical insight yielded by the current study was that the NSCs localized primarily to the peritumoral stroma, with limited penetration into the tumor parenchyma (Fig. 3E). Thus, it is possible that improving the delivery of a drug to tumor nodules using NSCs may not be sufficient to improve its efficacy if other parameters dominate the response to treatment. For example, NSC-mediated drug delivery only improved the therapeutic efficacy of PTX (15) but not cisplatin (unpublished data). Other important parameters include: diffusion limits [cisplatin (200 cell layers) vs PTX (80 layers)(36)]; peritoneal-to-plasma area under the concentration-time curve ratio [cisplatin (7.8-21) (37,38) vs PTX (853) (39)]; treatment schedule [cisplatin (slow release, treated weekly) vs PTX (burst release, treated bi-weekly); impaired biological activity of cisplatin after release from NSCs, microenvironmental priming by PTX that improves drug penetration or immune stimulation upon repeated dose cycles (40–44). It will be important to consider and address these factors when selecting and developing therapeutic cargo for effective NSC delivery. Conversely, co-localization of NSCs to the tumor stroma provides an advantage in targeting the tumor stroma components that support tumor growth and metastasis(45). This can also be exploited to deliver immunotherapeutic reagents in a targeted setting, preventing systemic disadvantages of immunotherapy.
NSC Safety Considerations: Our result shows that HB1.F3.CD21 NSCs are non-tumorigenic in the peritoneal setting (Fig. 4C), consistent with our current clinical data in the glioma setting, as patients in phase I trials of allogeneic NSC-mediated enzyme/prodrug and CRAd-S-pk7 treatments have tolerated multiple intracranial administrations without adverse events or evidence of secondary tumorigenicity (16). In stark contrast, MSCs have been reported to functionally engraft into peritoneal organs (46,47), and can promote ovarian tumor growth by inducing the expression of IL-1, associating with macrophages, and transforming into carcinoma-associated MSCs (48). IP MSC administration has also been shown to increase pro-inflammatory cytokines in mice, triggering such dramatic omental immune cell influx that the organ doubled in weight (49). The results presented here show that NSCs induce negligible immunological recognition in vitro (Fig. 4A); however, further investigation of the potential immunomodulatory effects of NSCs within the IP cavity will be important within the context of future therapeutic efficacy studies, particularly for repeated NSC administrations.
Although autologous mesenchymal stem cells have also been used clinically to improve viral delivery to ovarian metastases (12), our allogeneic, off-the-shelf cell NSC line has essential practical advantages that enable cost-effective scale-up and greater reproducibility between patients. Following extensive characterization of the HB1.F3.CD NSC line, (50) we have already pioneered the clinical translation of genetically modified NSCs for four cancer (glioma and neuroblastoma) therapies (51). We have experience expanding, modifying, and banking these NSCs as “off-the shelf” products, readily available for large trials at multiple sites. As NSC-based therapies progress into the clinic for targeted cancer treatment within the peritoneal setting, understanding the pharmacokinetics of the NSCs administered into this setting is important prior to performing IND-enabling studies involving NSCs modified with a therapeutic. Our plan is to streamline the translation of our existing therapeutic approaches to first-in-human phase I trials for stage III ovarian cancer patients who fail surgical and chemotherapy standard of care, taking advantage of the Good Manufacturing Practices (GMP) standard operating procedures (SOPs) established for our ongoing clinical studies in other cancer settings.