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Hirschberg et al. Neuroimmunol Neuroinflammation 2018;5:27 I http://dx.doi.org/10.20517/2347-8659.2018.31 Page 7 of 11

PDT of F98 rat glioma cells ex vivo. AlPcS2a is a photosensitizer which enters the cell by endocytosis, and
tends to localize in endosomes and lysosomes. PDT at relatively low light dose rates causes partial damage to
lysosomes resulting in the release of hydrolases, which trigger both apoptotic and/or autophagy cell death.

Fischer rats and F98 (syngeneic) and BT C (allogeneic) glioma cells were used in these experiments, in an
4
[62]
in vivo brain tumor development model . Co-incubation of naive Ma with AlPcS2a-PDT treated F98 glioma
cells led to pronounced morphological changes of the Ma. Naïve Ma were round in shape, approximately
10 to 15 μm in diameter and are composed of an equal population of both adherent and floating cells in vitro.
By contrast, activated Ma were significantly larger, irregular in shape with increased intracellular inclusions
and all of the cells were adherent in culture. Inoculation of these primed Ma (acting as APC), significantly
inhibited but did not completely prevent the growth of F98-induced tumors in the brain. Complete
suppression of tumor development though, was obtained via AlPcS2a-PDT-treated tumor cell primed Ma
inoculation combined with direct intra-cranial injection of allogeneic glioma cells. Interestingly, allogeneic
glioma cells injected into the brain in one hemisphere did not form tumors but surprisingly slowed the
growth of syngeneic tumors induced in the contra-lateral hemisphere in the same animal. This appeared
to indicate a systemic immune response generated via i.c inoculation by allogeneic glioma cells, though
inadequate to prevent tumor development, did have an inhibiting effect.

Allogeneic cells likely contain antigen determinants shared with the syngeneic tumor, leading to the
observed reduction in tumor growth. This hypothesis is in agreement with the previous findings of
Stathopoulos et al. [63,64] in preclinical studies in rats using both allo and syngeneic stimulation. The
underlying DAMPs developed by AlPcS2a-PDT, as has been previouslydemonstrated for Hyp-PDT [54-58] ,
remains to be determined in detail.

In vivo produced vaccines
In all of the above mentioned studies glioma tumor cells were PDT treated in vitro. In a subset of brain
[65]
tumor patients, harboring surgically inaccessible tumors, interstitial PDT (iPDT) has been evaluated . Here
light treatment is applied via stereotactically placed implantable fibers directly into the tumor. iPDT could be
combined with direct injection of naïve APCs as has been done in several previously described extra-cranial
experimental tumor models [26-28] . This protocol translated to intra-cranial tumors is illustrated in Figure 3
and is presently under development.

This combined iPDT-APC injection strategy would both directly destroy portions of the tumor and
additionally induce in situ APC activation which enhances antigen acquisition and processing as well as
migration of the APCs to draining lymph nodes. This in vivo produced vaccine would potentially enhance
the primary PDT effect and prevent tumor regrowth. It would also obviate the time consuming and costly
necessity of priming APCs in vitro.

CONCLUSIONS
Although the experience with PDT/PCI produced anti HGG vaccines is limited and no clinical trials have
been done, PDT/PCI appears to be an inducer of immunogenic cancer cell death, an important step in
the afferent phase of the immune anti-tumor response. Light activated induced immunotherapy therefore
holds the potential to become a complementary therapeutic option for for patients with HGG. Taking into
account the penetration limitations of light activated therapies in the brain the further development of ex
vivo PDT/PCI generated APC or peptide vaccines seems the most attractive approach. A deeper and detailed
understanding of the induction of the antitumor immunity induced by light activated therapies would allow
in the defining of protocols which would focus and enhance the immune system to recognize and prevent
the inevitable post-operative recurrence of the tumor. Combining PDT induced anti-tumor vaccines with
other therapeutic modalities including check-point inhibitors, is an exciting field to explore. Although