"Colon cancer cell treatment with rose bengal generates a protective immune response via immunogenic cell death"

Article source link is here.

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"Abstract:

Immunotherapeutic approaches to manage patients with advanced gastrointestinal malignancies are desired; however, mechanisms to incite tumor-specific immune responses remain to be elucidated. Rose bengal (RB) is toxic at low concentrations to malignant cells and may induce damage-associated molecular patterns; therefore, we investigated its potential as an immunomodulator in colon cancer. Murine and human colon cancer lines were treated with RB (10% in saline/PV-10) for cell cycle, cell death, and apoptosis assays. Damage-associated molecular patterns were assessed with western blot, ELISA, and flow cytometry. In an immunocompetent murine model of colon cancer, we demonstrate that tumors regress upon RB treatment, and that RB induces cell death in colon cancer cells through G2/M growth arrest and predominantly necrosis. RB-treated colon cancer cells expressed distinct hallmarks of immunogenic cell death (ICD), including enhanced expression of calreticulin and heat-shock protein 90 on the cell surface, a decrease in intracellular ATP, and the release of HMGB1. To confirm the ICD phenotype, we vaccinated immunocompetent animals with syngeneic colon cancer cells treated with RB. RB-treated tumors served as a vaccine against subsequent challenge with the same CT26 colon cancer tumor cells, and vaccination with in vitro RB-treated cells resulted in slower tumor growth following inoculation with colon cancer cells, but not with syngeneic non-CT26 cancer cells, suggesting a specific antitumor immune response. In conclusion, RB serves as an inducer of ICD that contributes to enhanced specific antitumor immunity in colorectal cancer."

"Discussion:

...In the current study, we have provided several lines of evidence demonstrating that RB exhibited pronounced direct cytotoxicity in colorectal cancer cells both in vivo and in vitro. Our data also confirmed RB-induced prominent cell growth arrest at the G2/M phase and predominant necrotic cell death that was partially dependent on lysosome function. Furthermore, the findings from the current study identified that RB promoted expression of hallmarks related to ICD in colon cancer cell lines. Vaccination with RB-treated colon cancer cells and intralesional tumor injection resulted in retardation in tumor growth or prevention of subsequent tumor formation following challenge with the same tumor cells. These findings show that RB may serve as an inducer of ICD that contributes to enhanced specific antitumor immunity in colorectal cancer. Additional studies are warranted to elucidate the therapeutic potential of RB-induced ICD."

Could PV-10 (Rose Bengal) be implicated in different kinds of cell death?

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Reference article: Garg et al., Immunogenic versus tolerogenic phagocytosis during anticancer therapy: mechanisms and clinical translation, Cell Death and Differentiation (2016) 23, 938–951.

N.B. There is no reference to Rose Bengal (generic name) or PV-10 (proprietary name) in the February 2015 Garg et al. article.

Article Abstract: Phagocytosis of dying cells is a major homeostatic process that represents the final stage of cell death in a tissue context. Under basal conditions, in a diseased tissue (such as cancer) or after treatment with cytotoxic therapies (such as anticancer therapies), phagocytosis has a major role in avoiding toxic accumulation of cellular corpses. Recognition and phagocytosis of dying cancer cells dictate the eventual immunological consequences (i.e., tolerogenic, inflammatory or immunogenic) depending on a series of factors, including the type of ‘eat me’ signals. Homeostatic clearance of dying cancer cells (i.e., tolerogenic phagocytosis) tends to facilitate pro-tumorigenic processes and actively suppress antitumour immunity. Conversely, cancer cells killed by immunogenic anticancer therapies may stimulate non-homeostatic clearance by antigen-presenting cells and drive cancer antigen-directed immunity. On the other hand, (a general) inflammatory clearance of dying cancer cells could have pro-tumorigenic or antitumorigenic consequences depending on the context. Interestingly, the immunosuppressive consequences that accompany tolerogenic phagocytosis can be reversed through immune-checkpoint therapies. In the present review, we discuss the pivotal role of phagocytosis in regulating responses to anticancer therapy. We give particular attention to the role of phagocytosis following treatment with immunogenic or immune-checkpoint therapies, the clinical prognostic and predictive significance of phagocytic signals for cancer patients and the therapeutic strategies that can be employed for direct targeting of phagocytic determinants.

Provectus says PV-10/Rose Bengal:

• Does not rely on a single pathway to work [I assume 'signalling pathway'],

• Does not focus on a single receptor to work [I assume 'cell receptor'], and

• Has no known resistance [I assume little no cancer drug resistance].

A highly specific compound in its targeting of only diseased (cancerous) tumors/lesions, tissue and cells, sparing healthy ones in the process, might the veracity of PV-10/Rose Bengal's "multiplicity" be based in its lack of "specificity" in regards to cell death?

That is, might Provectus' investigational compound's apparent implication in different kinds of cell death help explain why PV-10/Rose Bengal does not rely on a single pathway or focus on a single receptor to work, and has no known resistance?

Of note in Garg et al.'s article are (a) a table describing "major cell death pathways and their immunobiological" profiles and (b) a figure illustrating "therapeutic exploitation of phagocytosis of dying cancer cells for T-cell-mediated cancer cell elimination."

The table of major cell death pathways includes (i) apoptosis, (ii) autophagy (autophagic cell death) and (iii) immunogenic cell death.

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The illustration appears to draw three paths to anti-tumor immunity; one that is direct (e.g., DAMPs like HMGB1), and two that additionally employ co-stimulatory signals such as TLR agonists or co-inhibitory signals such as immune checkpoint therapy.

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A sampling of Provectus and independent medical researcher work implicates PV-10 in apoptosis, autophagy, necrosis, and immunogenic cell death:

• Provectus: "The patient who had chemotherapy radioresistant recurrent disease in the head and neck area had rapid necrosis of the injected metastasis and this as well as 3 uninjected sc metastases underwent complete remission over a period of several months," "Rose Bengal induces dual modes of cell death in melanoma cells and has clinical activity against melanoma," Melanoma Research 16(Suppl 1):S8, Sep 2006

• Provectus: "PV-10 accumulation elicits acute autophagy of cancer cells," "Chemoablation of Metastatic Melanoma with PV-10," Presentation at 4th Interdisciplinary Melanoma & Skin Cancer Centres Meeting, Sydney, AU, Nov 2010

• University of Illinois at Chicago: "Treatment of colon cancer cells with PV-10 induced cell cycle arrest, apoptosis, autophagy, and significant ER stress; consistent with immunogenic apoptosis," "PV-10 Induces Potent Immunogenic Apoptosis in Colon Cancer Cells," ASC 2016

• Quote by Provectus about Moffitt Cancer Center work: "This paper is a watershed event in the development of PV-10, walking the reader through all the steps of immune activation after PV-10 injection, from immunogenic cell death and signaling via release of HMGB1, dendritic cell recruitment and infiltration into draining lymph nodes, activation of tumor-specific T cells, and killing of uninjected tumors upon infiltration by these T cells." "Intralesional Rose Bengal in Melanoma Elicits Tumor Immunity Via Activation of Dendritic Cells by the Release of High Mobility Group Box 1," Oncotarget 2016.

Garg et al. observe "The mechanisms of cancer cell death elicited by anticancer therapy and the type of phagocytes (e.g., tumour-resident versus therapy-recruited) interacting with dying cells are decisive factors in making a difference between anti-inflammatory or pro-inflammatory responses."

Some cell death via PV-10 occurs in the injected lesion or tumor (i.e., tumor-resident), which is the upstream trigger of subsequent cell death via a tumor-specific immune response (i.e., therapy-recruited).

PV-10 is an immunotherapy

Click to enlarge. Original image source (Google search of "immunotherapy").
Colored editing above is mine.

I think the upshot of the University of Illinois at Chicago's (UIC's) abstract at the 2016 annual meeting of the Academic Surgical Congress -- PV-10 Induces Potent Immunogenic Apoptosis in Colon Cancer Cells -- is its contribution to the growing mound of preclinical and clinical data that:

• First, PV-10 (Rose Bengal) ablates all solid tumors (UIC: "We have previously demonstrated that human and murine colon cancer cells undergo near complete cell death in vitro and in vivo upon direct exposure to PV-10, a synthetic dye currently in clinical trails for intralesional therapy of in-transit melanoma", and

• Second, PV-10 primes the immune system in all solid tumors (UIC: "Therefore, based on these results, further evaluation of PV-10 as a potential agent to stimulate immunologic cell death in solid tumors is warranted.").

Said differently, and perhaps with the benefit of more clinical data, particularly that from Provectus' Phase 1b/2 study PV-10 in Combination With Pembrolizumab for Treatment of Metastatic Melanoma, PV-10 should be called an immunotherapy. This small molecule that generates clinical benefit for and positive outcomes in cancer patients from its physical chemistry has feelings too.

Moffitt Cancer Center in its SITC 2015 poster entitled Intralesional Rose Bengal in Melanoma Elicits Tumor Immunity via High Mobility Group Box 1, noted:

"IL RB therapy also increased antigen-specific T cell proliferation and enhanced tumor regression. In addition, IL RB facilitated dendritic cells (DCs) infiltrating lymph nodes draining from tumor. Incubation of melanoma cells with RB led to necrosis and the release of High Mobility Group Box 1 (HMGB1), which activated DCs. The blockade of HMGB1 significantly reduced the antigen-presenting ability of DCs."

UIC noted in their ASC 2016 abstract:

"Treatment of colon cancer cells with PV-10 induced cell cycle arrest, apoptosis, autophagy, and significant ER stress; consistent with immunogenic apoptosis. In order for cytotoxic agents to act as potential immunotherapeutic strategies in the treatment of solid tumors, immunogenic cell death targeting the endoplasmic reticulum (ER), leading to ER stress may be critical."

Underlined emphasis above and below is mine. 

Playing scientist using Google, note for example abstract comments from Zhu et al. in Endoplasmic reticulum stress and its regulator XBP-1 contributes to dendritic cell maturation and activation induced by high mobility group box-1 protein (Int J Biochem Cell Biol. 2012 Jul;44(7):1097-105):

"High mobility group box-1 protein (HMGB1) had been proved to induce maturation and activation of dendritic cell (DC), however, the endogenous changes and mechanisms underlying are unknown. Since endoplasmic reticulum stress (ERS) activates an adaptive unfolded protein response (UPR) that facilitates cellular survival and repair, we hypothesized that HMGB1 may regulate the function of DC by modulating ERS. In our study, HMGB1 stimulation induced significant ERS responses in DCs in a time- and dose-dependent manner, demonstrated by the up-regulation of a number of ERS markers."

Note: CGMP = Current Good Manufacturing Practice (CGMP). See for example Drug Applications and Current Good Manufacturing Practice (CGMP) Regulations.

provectus IS a t-cell company

H/t InvestorVillage poster bradpalm1: A research team from the University of Illinois at Chicago (UIC) published a paper in July entitled The Potential of Intralesional Rose Bengal to Stimulate T-Cell Mediated Anti-Tumor Responses (Maker et al., J Clin Cell Immunol 2015, 6:4). This same team previously published results of their murine model work and research on PV-10 for colon cancer. See June 5, 2015 blog post Intralesional Injection of Rose Bengal Induces an Anti-tumor Immune Response and Potent Tumor Regressions in a Murine Model of Colon Cancer.

Blog post takeaways:

• Rose Bengal (PV-10)/PV-10 (Rose Bengal) induces/generates/creates/leads to anti-tumor immunity. To some the so-called Holy Grail of cancer therapy is achieving durable [system- or body-wide] anti-tumor immunity in a cancer patient.

• PV-10 can prime the immune system (through the direction injection of the compound into cancerous lesions), and help it defeat distant cancerous lesions around the body.

• The compound exposes antigens to the immune system that otherwise would not be exposed, or that otherwise would escape detection by (and thus presentation to) the immune system.

• PV-10 can help with T cell generation & circulation, even after treatment with immune checkpoint inhibitors fails.

• UIC's work reproduced Moffitt Cancer Center's (Moffitt's) work, which reproduced Provectus' founders' work.

• UIC's work that contributed to their paper was funded in part by a grant (non-specific to Provectus and PV-10) from the National Institutes of Health.

Recall that Provectus has a two-prong approach to fighting (killing) cancer: (i) local effect/tumor ablation (anti-tumor cytotoxicity) and (ii) systemic effect/tumor-specific immune response (induction of anti-tumor immunity)

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UIC and Moffitt independently reproduced the veracity of both prongs:

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UIC's paper reviews PV-10's agnosticity in destroying tumors into which it is injected (i.e., anti-tumor cytotoxicity):

• Melanoma,
• Breast cancer,
• Ovarian cancer,
• Gastric cancer, and
• Sarcoma.

The paper discusses PV-10's agnosticity in generating anti-tumor immunity resulting from local tumor injection (i.e., from resultant anti-tumor cytotoxicity):

• Melanoma,
• Breast cancer, and
• Hepatocellular carcinoma (HCC).

The UIC team appears to have conducted murine model work on PV-10 in HCC, noting "Our current research is establishing the role of RB in generating anti-tumor immune responses in gastrointestinal cancer and liver metastases:"

"Furthermore, in syngeneic orthotopic models of murine hepatocellular carcinoma (HCC), RB similarly induced chemoablation in all treated tumors. Twenty-one to 81 days after RB treatment, when re-challenged with the same tumor, durable immunity was demonstrated in 14/14 animals without measurable tumor formation, whereas B16-F10 melanoma tumors, i.e., non-HCC cells, were able to be established in 13/13 animals [20]. Additionally, immunity to establishment of a new HCC tumor could be created through adoptive transfer of splenocytes from treated animals. This was an interesting finding given that analysis of splenic composition from animals with B16 melanoma that experienced distant lung tumor regression after RB treatment of flank tumors did not demonstrate any difference in the percent of T cells, Tregs, NK cells, B cells, myeloid derived suppressor cells, or macrophages [15]. The authors further demonstrated that bystander lesions disappeared or decreased in size in HCC models, whereas, no bystander tumors were ever observed to resolve in nude mice without a competent T-cell immune system. These experiments established that, similar to melanoma, in immunocompetent mice with orthotopic primary hepatocellular carcinoma flank tumors, an anti-tumor response can be induced by priming the animals with RB treatment of tumors. These experiments raise the possibility that RB induced cytotoxicity exposes antigens and mounts an immune response that may protects animals from additional tumor formation that can be adoptively transferred to other animals using splenocytes." {Underlined and bolded emphasis is mine}

Speaking of antigens, it's all about the antigens:

"These experiments established that in human and murine melanoma and breast cancer, there appeared to be an antigen-driven T-cell response that has the potential to activate T-cells and impart antitumor responses in bystander lesions and distant metastases."

Why are antigens important? Because they facilitate the priming of the immune system through their presentation to it:

"These findings may imply that tumor response in the primary lesion may be able to prime the immune system of patients for activity against distant lesions."

The UIC team's conclusions could be profound, among them:

• "RB-mediated tumor cell death may expose tumor antigens that may otherwise evade immune detection."

• "Though most profoundly described in melanoma cells, clearly an immunoreactive malignancy, the effect has been shown to be not limited to one specific type of malignancy. 

• "Decrease in tumor burden and stimulation of an immune response with PV-10 has been demonstrated in animal models of metastasis, and correlations of these responses in clinical studies is consistent with such results." Provectus' CTO Dr. Eric Wachter, PhD has said in a 2013 white paper: "PV-10 murine research demonstrated unambiguously, Dr. Wachter noted, that tumor burden is a critical variable in predicting response to a combination therapy." See Radiation (September 22, 2015) on the blog's Current News page.

• "That PV-10 treatment can potentially increase circulating cytotoxic T-cells, even in patients who were previously treated with immune-activating checkpoint blockade, supports the possibility that RB induced cytotoxicity may activate T-cells that are responsible for the bystander effect on untreated lesions."

• As such, intralesional therapy with RB may be a promising new mode of therapy to stimulate T-cell mediated anti-tumor immune responses."

UIC's work was conducted in part under an NIH grant.

Intralesional Injection of Rose Bengal Induces an Anti-tumor Immune Response and Potent Tumor Regressions in a Murine Model of Colon Cancer

An abstract from the March 2015 Society of Surgical Oncology 68th Annual Cancer Symposium by Pardiwala et al., University of Illinois at Chicago (P134, see page S86):

INTRODUCTION: Early phase studies using intratumoral injection of PV-10 (10% Rose Bengal) have shown regression of in-transit melanoma deposits and non-treated bystander lesions. The effects of PV-10 on colorectal cancer (CRC) cells and established tumors is unknown. METHODS: Murine and human colorectal cancer cells were treated in vitro with varying concentrations of PV-10. Cell viability was determined by the MTS assay, trypan blue, DAF-FM, and SNARF-1 staining. To determine an underlying immune mechanism, a murine CT26 syngeneic CRC model was utilized. Bilateral subcutaneous CRC cell tumors were established in Balb/C mice and one tumor in each mouse was injected with PV-10 at a dose equal to half the calculated tumor volume. Tumors were measured daily. Splenocytes from treated animals were co-cultured with irradiated CT26 cells and supernatants analyzed for INF-γ production by ELISA. RESULTS: PV-10 induced near total cell death, corresponding increases in nitric oxide production, and decreased intracellular pH in both CT26 murine and HT29 human CRC cells within hours of exposure compared to controls (p<0.01), and at levels similar to 5FU. Treatment of subcutaneous tumors with a single injection of intralesional PV-10 led to near complete responses in all animals within days of exposure and significant regression of the injected lesions compared to controls (n=6 per group, p=0.027). PV-10 treatment was associated with occasional bystander responses in contralateral untreated tumors and trended towards a decreased rate of growth in these lesions. Splenocytes isolated from tumor bearing mice treated with PV-10 displayed enhanced tumor-specific IFN-γ production compared to splenocytes from PBS-treated mice (p = 0.025). CONCLUSION: Rose Bengal induced potent cell death in human and murine colon cancer cells in vitro. Intralesional injection in established tumors induced an anti-tumor immune response and significant tumor regressions in vivo. These studies establish that intralesional PV-10 therapy warrants further study as a potential immunotherapeutic agent in colorectal cancer and metastases.

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