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Page 6 of 15 Gururangan et al. Neuroimmunol Neuroinflammation 2018;5:45 I http://dx.doi.org/10.20517/2347-8659.2018.44

[40]
immune gene signatures . In keeping with these gene signatures, group B recurrent tumors had higher
infiltration of CD4+ and CD8+ T cells. When group A and group B tumors from diagnosis were evaluated
for secretion of immune cytokines following stimulation, group B tumors secreted higher amounts of TNF-α
[40]
(2.7 fold), IFN-γ (5.3 fold), and granulocyte-macrophage colony-stimulating factor (GM-CSF) (5.1 fold) .
In contrast to ependymomas, the tumor microenvironment of diffuse pontine glioma, a midline tumor
with a dismal prognosis, is predominantly populated by CD11b+ macrophages which contain scant CD3+
T-lymphocytes; moreover, in contrast to malignant glioma, diffuse intrinsic pontine glioma ex-vivo cell
[41]
cultures (patient-derived) release significantly less cytokines .

TtRNA DC vaccines in pediatric brain tumors
DC vaccines used in cancer are of four main categories and include peptide vaccines, cellular vaccines
(tumor or immune cells), viral vector vaccines, and nucleic acid (DNA and RNA) vaccines [42,43] . Our lab
has shown the use of RNA-pulsed DCs to be a versatile platform for activating tumor-specific T cells
in vitro and in vivo in several murine and human systems. Several clinical trials have been conducted
demonstrating the feasibility and safety of tumor lysate or RNA-pulsed DCs in human patients [44-46] . While
specific tumor-associated antigens such as carcinoembryonic antigen, telomerase reverse transcriptase,
melanoma antigens, epidermal growth factor receptor variant III (EGFRvIII), and human cytomegalovirus
(CMV) phosphoprotein 65 (pp65) have all been successfully utilized in trials as either peptide vaccines
or RNA-encoded antigens in DCs [46-48] , studies have demonstrated that the majority of endogenous anti-
[49]
tumor immune responses in patients with malignancy are against unidentified, patient-specific antigens .
While use of ttRNA pulsed DCs to expand tumor-specific lymphocytes allows for these patient-specific
antigens to be targeted, sufficient tumor tissue for clinical-scale vaccination is not always readily available.
Our laboratory has utilized amplification of ttRNA with reverse-transcriptase primed polymerase chain
reaction to generate cDNA library templates encoding for the antigenic content of tumor cells from as few
as 500 starting tumor cells. Through inclusion of a T7 RNA polymerase binding site in the 5’ primer used
for amplification, ttRNA can be readily generated through in vitro transcription after cDNA amplification.
Using such techniques, we have been able to generate enough RNA for clinical DC vaccine preparations
from colorectal tumors, renal carcinoma, and pediatric and adult brain tumor specimens using excess tumor
material harvested during surgical resection [50-52] .

While expansion of tumor cells using in vitro culture is feasible, primary brain tumor cells are often
difficult to propagate and gene expression microarray analysis has demonstrated that most tumor specific
genes expressed in vivo are not recapitulated within in vitro propagated tumor cells. Furthermore, we have
demonstrated in murine intracranial glioma models that a significant shift in brain tumor gene expression
[53]
is induced in response to host anti-tumor immunity . This strongly suggests that the antigenic content
of tumor cells propagated in vitro will be significantly different than in vivo propagated tumors, and thus
the relevance of in vitro propagated tumors as an antigenic source for immunotherapy is questionable.
This observation prompted us to investigate the capacity to amplify the RNA content of tumor cells
isolated directly from surgically resected malignant glioma specimens to utilize an antigenic source more
representative of the antigens expressed within patients’ tumor cells in vivo. Based on current clinical
[54]
protocols utilizing ttRNA pulsed DCs , it is possible to produce up to 750 µg of amplified tumor mRNA
per patient. We have successfully amplified tumor mRNA to clinical scale (over 1mg) from as few as
500 astrocytoma cells from resected human glioma specimens from adult and pediatric brain tumors.
Enrichment of tumor antigens can be done using subtractive hybridization of excess pooled normal brain
RNA from tumor RNA prior to amplification and in vitro RNA synthesis and verification of enrichment of
tumor-associated genes by comparative real-time PCR.

Once enough ttRNA is obtained, it can then be introduced via electroporation (300 V for 500 μsecs) into
immature DCs derived from patient derived peripheral blood monocytes obtained via a peripheral blood
mononuclear cell (PBMC) collection via apheresis. Differentiation of monocytes into DCs is achieved in in vitro