**1. Introduction**

The rodent H-1 protoparvovirus (H-1PV) (for an overview of H-1PV classification and biology, we redirect the readers to a recent review by Bretscher and Marchini [1]) was first discovered as a contaminating agent in xeno-transplanted human tumor cell lines [2]. Originally identified as a pathogen, which lethally affects rat fetuses and newborn rats by causing cerebellar hypoplasia and hepatitis [3], H-1PV was later found to preferentially replicate in rat- and in human-transformed or tumor-derived cell cultures, while sparing their non-malignant counterparts [4,5]. H-1PV intrinsic oncotropism and oncoselectivity are a complex phenomenon based on multiple molecular determinants, which are underrepresented in normal cells, but characteristic of tumor cells [6]. Importantly, humans are not naturally infected with this virus, and no association between H-1PV and human disease has been observed [7]. Two early clinical studies of virus administration to cancer patients—dating back to the 1960s and 1990s of last century—demonstrated the lack of H-1PV pathogenic effects and the feasibility of the approach [8,9], thus laying the groundwork for the development of parvovirus (PV)-based oncolytic virotherapy. Three decades of laboratory efforts brought about extensive preclinical evidence of H-1PV broad tumor-suppressive potential [5,10]. Furthermore, it became increasingly apparent that in addition to directly inducing cancer cell death (oncolysis), H-1PV was also capable of exerting immuno-stimulating effects in various preclinical cancer models [11,12].

PV induced immune system stimulation results from multiple infection-associated immunogenic events. Depending on the tumor model, virus dose, route of administration and the immunological status of the host, one or another immunogenic stimulus may prevail [11]. Regardless of the particular mechanism involved, PV-mediated immunomodulation contributes to the "warming up" of the tumor microenvironment (TME) (Figure 1), increases tumor visibility and enhances immune cell reactivity [13]. H-1PV infectionassociated immunogenic events and their impact on the immune system are reviewed in detail elsewhere [12,13], and briefly summarized below.


inhibits the growth of lymphatic endothelium-derived tumors (Kaposi's sarcoma). Furthermore, recombinant propagation-deficient parvoviral vectors armed with angiostatic chemokines achieve significant reduction of vascular endothelial growth factor (VEGF) expression in Kaposi's sarcoma cells [19]. Given the control exerted by the vasculature of tumors over their infiltration with immune cells, these effects are likely to contribute to H-1PV immuno-stimulating activity, as further discussed below. Altogether, these data warrant validation of H-1PV as a tool against highly vascularized cancers, e.g., glioblastoma, one of the most angiogenic human tumors. cells sustain an abortive H-1PV infection in vitro. In animal models, virus treatment inhibits the growth of lymphatic endothelium-derived tumors (Kaposi's sarcoma). Furthermore, recombinant propagation-deficient parvoviral vectors armed with angiostatic chemokines achieve significant reduction of vascular endothelial growth factor (VEGF) expression in Kaposi's sarcoma cells [19]. Given the control exerted by the vasculature of tumors over their infiltration with immune cells, these effects are likely to contribute to H-1PV immuno-stimulating activity, as further discussed below. Altogether, these data warrant validation of H-1PV as a tool against highly vascularized cancers, e.g., glioblastoma, one of the most angiogenic human tumors.

• H-1PV impact on tumor vasculature: It has been demonstrated that endothelial (precursor) cells may constitute direct targets for parvovirus-mediated toxicity. These

*Cancers* **2021**, *13*, x 3 of 15

**Figure 1.** H-1PV-induced modulation of tumor microenvironment immune landscape. (**a**) Immunosuppressive ("cold") tumor microenvironment (TME) of a solid tumor. The tumor is often infiltrated with abundant immunosuppressive regulatory T cells (Treg)/myeloid-derived suppressor cells (MDSC). Tumor-infiltrating lymphocytes (TILs) (CD8+ CTLs, CD4+ Th cells) are scarce and/or anergic. Tumor and various TME cells produce anti-inflammatory cytokines to maintain immune suppression and facilitate tumor growth and dissemination. (**b**) Tumor infection with H-1PV results in immunogenic tumor cell death leading to the release proinflammatory cytokines, pathogen- and danger-associated molecular patterns (PAMPs and DAMPs), which alarm the immune system. The infection of tumor cells does not necessarily have to be productive for this immuno-stimulating effect to be achieved. Furthermore, abortive infection of immunocytes (CTLs, Th cells, monocytes/macrophages) with H-1PV can also lead to their activation. In contrast, H-1PV inhibits the immune suppressive functions of Treg cells. An immunological switch takes place and converts the "cold" TME into a "warmed up" (inflamed) one. Virus-mediated immuno-conversion of TME favors the mounting of enhanced antitumor immune responses. **Figure 1.** H-1PV-induced modulation of tumor microenvironment immune landscape. (**a**) Immunosuppressive ("cold") tumor microenvironment (TME) of a solid tumor. The tumor is often infiltrated with abundant immunosuppressive regulatory T cells (Treg)/myeloid-derived suppressor cells (MDSC). Tumor-infiltrating lymphocytes (TILs) (CD8+ CTLs, CD4+ Th cells) are scarce and/or anergic. Tumor and various TME cells produce anti-inflammatory cytokines to maintain immune suppression and facilitate tumor growth and dissemination. (**b**) Tumor infection with H-1PV results in immunogenic tumor cell death leading to the release proinflammatory cytokines, pathogen- and danger-associated molecular patterns (PAMPs and DAMPs), which alarm the immune system. The infection of tumor cells does not necessarily have to be productive for this immuno-stimulating effect to be achieved. Furthermore, abortive infection of immunocytes (CTLs, Th cells, monocytes/macrophages) with H-1PV can also lead to their activation. In contrast, H-1PV inhibits the immune suppressive functions of Treg cells. An immunological switch takes place and converts the "cold" TME into a "warmed up" (inflamed) one. Virus-mediated immuno-conversion of TME favors the mounting of enhanced antitumor immune responses.

> The above-outlined H-1PV potential for creating a proinflammatory immune environment and alerting the immune system to the presence of a tumor opens prospects for combining the virus with various immunomodulators or other therapeutic agents endowed with immuno-stimulating properties. This combinatorial approach is in particular promising for the treatment of human tumors that remain presently incurable and pose continuing research and clinical challenges. Pancreatic ductal adenocarcinoma (PDAC), glioblastoma, colorectal cancer (CRC) and melanoma are among those cancers, which are The above-outlined H-1PV potential for creating a proinflammatory immune environment and alerting the immune system to the presence of a tumor opens prospects for combining the virus with various immunomodulators or other therapeutic agents endowed with immuno-stimulating properties. This combinatorial approach is in particular promising for the treatment of human tumors that remain presently incurable and pose continuing research and clinical challenges. Pancreatic ductal adenocarcinoma (PDAC), glioblastoma, colorectal cancer (CRC) and melanoma are among those cancers, which are urgently calling for novel therapeutic strategies. H-1PV-based immunotherapeutic combinations are reviewed below, which aim at targeting these devastating malignancies.
