Adenovirus vectors with modified tropism for the treatment of colorectal cancer

Despite the rapid advance of cancer research and improvement of conventional therapeutic regimes like chemotherapy, surgery, immunotherapy or radiotherapy, cancer remains a leading cause of death worldwide. Metastatic disease normally represents the most deadly stage and leads to the poorest prognosis. The disseminated location of metastases makes it even more difficult nowadays for existing drugs to target tumors without damaging healthy tissues. The work presented in this Doctoral Thesis is aimed at developing targeted adenoviral vectors and regimes that can be applied to the treatment of cancer, especially colorectal cancer. Gene therapists have explored widely interactions of the viruses to cancer and normal cells and have proved that molecular modifications in the capsid can unleash striking differences in viral tropism. In this Doctoral Thesis, the utility of arginine-glycine-aspartic acid (RGD) targeting αvβ integrins substituted for the lysine-lysine-threonine-lysine (KKTK) domain of the fiber shaft or inserted in the HI-loop of adenovirus serotype 5 (Ad5) was evaluated for increased tumor targeting and antitumor efficacy. Both modifications increased gene transfer efficacy in colorectal cancer cell lines and improved the tumor to-normal ratio after systemic administration of the vector. Furthermore, antitumor potency was not compromised with RGD modified viruses suggesting that an increased safety profile did not involve any loss of therapeutic effect. Treatments based on adenovirus vectors should not have negative effects on tumor progression or metastases. In order to evaluate this possibility, we designed a novel murine model of human colorectal cancer (CoCa) to test our treatments. To this end, we have developed a readily imageable mouse model of colorectal cancer featuring highly reproducible formation of spontaneous liver metastases derived from intrasplenic primary tumors. We optimized several experimental variables, and found that the correct choice of cell line and genetic background of the recipient mice as well as their age, were critical for the establishment of a useful animal model. A magnetic resonance imaging (MRI) protocol was optimized for use with this mouse model, and demonstrated to be a powerful method for analyzing the antitumor effects of an experimental therapy. Poor spreading of the virus through tumor tissue is one of the major issues limiting efficacy of oncolytic adenoviruses, even after local administration by intratumoral injection. In this study, ECM-degrading proteases relaxin, hyaluronidase, elastase, and macrophage metalloelastase (MME) were used to increase oncolytic adenovirus spreading. Moreover, MME improved the overall antitumor/antitumour efficacy of oncolytic adenovirus in subcutaneous HCT116 xenografts. In a liver metastatic colorectal cancer model, intratumoral treatment of HT29 primary tumors with MME monotherapy or with oncolytic adenovirus inhibited tumor growth. Combination therapy showed no increased mortality in comparison to monotherapies. In addition, our work demonstrated for the first time in a metastatic animal model that MME, as a monotherapy or in combination with an oncolytic virus, does not increase tumor invasiveness. Co-administration of MME and oncolytic adenovirus may be a suitable approach for further optimization of metastatic colorectal cancer treatment. To summarise, we described how RGD moieties inserted in the fiber protein are capable of improving tumor targeting of wild-type or capsid-modified adenovirus vectors. We also showed that MME is a safe coadjuvant to be used in combination with oncolytic adenoviruses for intratumoral administration and we presented a highly optimized mouse model for liver metastasis of colorectal cancer.Syöpätutkimuksen nopeasta etenemisestä ja konventionaalisten hoitojen kehittymisestä huolimatta on syöpä edelleen yleisin kuolemaan johtava sairaus maailmassa. Syövän metastaasivaihe on usein vaikeimmin hoidettavissa ja paranemisennuste on huono. Metastaattisten syöpien hoito nykyisillä lääkkeillä on usein tehotonta ja lääkkeet saattavat vahingoittaa myös terveitä soluja. Tämän väitöskirjatyön tavoitteena oli kehittää syöpäsoluihin kohdennettuja terapeuttisia adenovirusvektoreita sekä niihin perustuvia syövän hoitomuotoja erityisesti kolorektaalisyövän hoitoon. Adenovirukset ovat vaipattomia viruksia joiden pintakerros, kapsidi, koostuu useista erilaisista proteiineista. Kapsidin rakenne ja koostumus vaikuttavat viruksen kykyyn infektoida erilaisia kudoksia ja soluja. Kapsidin keinotekoisella muokkauksella voidaan tehostaa viruksen infektiotehokkuutta tuumorisoluissa sekä heikentää viruksen kykyä infektoida terveitä soluja ja näin ollen parantaa adenovirusvektoreiden tehoa ja turvallisuutta syövän hoidossa. Tässä väitöstutkimuksessa olemme osoittaneet että voimme kohdentaa adenoviruksen infektiota tuumorisoluihin lisäämällä arginiini-lysiini-aspargiinihappo (RGD) domeenin tietyille alueille kapsidirakennetta. Selvitimme myös voisiko adenovirusvektoreihin perustuva syövän hoito muuttaa syöpäkudosta haitallisesti niin, että metastaasien todennäköisyys lisääntyy. Kehitimme tätä tutkimusta varten uudenlaisen ihmisen kolorektaalisyövän metastaasien muutoksia mittaavan hiirimallin. Tällä hiirimallilla voimme mitata tuumorin koon muutoksia sekä käytetyn terapeuttisen vektorin kykyä muuttaa syövän metastasointitaipumusta. Syövän hoitoon käytettävät adenovirusvektorit annostellaan usein intratumoraalisesti. Yksi suurimmista ongelmista adenovirusvektoreiden käytössä on terapeuttisten virusten tehoton leviäminen kaikkialle syöpäkudokseen. Tässä väitöstutkimuksessa tutkimme myös onkolyyttisen adenoviruksen ja matriksin metalloelastaasiproteiinin (MME) yhteisvaikutusta lisätä adenoviruksen kykyä levitä kaikkialle syöpäkudokseen. Väitöstutkimuksessa kuvattiin adenoviruksen kapsidin muokkaus joka kohdentaa viruksen paremmin syöpäkudokseen. Väitöstutkimuksessa osoitettiin myös MME:n olevan turvallinen adjuvantti annosteltuna samanaikaisesti terapeuttisten adenovirusten kanssa. Lisäksi väitöstutkimuksessa kehitettiin uusi ihmisen kolorektaalisyövän metastaasien muutoksia mittaavan hiirimalli

Optimized Mouse Model for the Imaging of Tumor Metastasis upon Experimental Therapy

Peer reviewe

Adenoviruses with an alpha(v)beta integrin targeting moiety in the fiber shaft or the HI-loop increase tumor specificity without compromising antitumor efficacy in magnetic resonance imaging of colorectal cancer metastases

Peer reviewe

MicroRNA-Mediated Suppression of Oncolytic Adenovirus Replication in Human Liver

Peer reviewe

Cancer-Associated Fibroblasts Neutralize the Anti-tumor Effect of CSF1 Receptor Blockade by Inducing PMN-MDSC Infiltration of Tumors.

Tumor-associated macrophages (TAM) contribute to all aspects of tumor progression. Use of CSF1R inhibitors to target TAM is therapeutically appealing, but has had very limited anti-tumor effects. Here, we have identified the mechanism that limited the effect of CSF1R targeted therapy. We demonstrated that carcinoma-associated fibroblasts (CAF) are major sources of chemokines that recruit granulocytes to tumors. CSF1 produced by tumor cells caused HDAC2-mediated downregulation of granulocyte-specific chemokine expression in CAF, which limited migration of these cells to tumors. Treatment with CSF1R inhibitors disrupted this crosstalk and triggered a profound increase in granulocyte recruitment to tumors. Combining CSF1R inhibitor with a CXCR2 antagonist blocked granulocyte infiltration of tumors and showed strong anti-tumor effects

Healthcare workers hospitalized due to COVID-19 have no higher risk of death than general population. Data from the Spanish SEMI-COVID-19 Registry

Aim To determine whether healthcare workers (HCW) hospitalized in Spain due to COVID-19 have a worse prognosis than non-healthcare workers (NHCW). Methods Observational cohort study based on the SEMI-COVID-19 Registry, a nationwide registry that collects sociodemographic, clinical, laboratory, and treatment data on patients hospitalised with COVID-19 in Spain. Patients aged 20-65 years were selected. A multivariate logistic regression model was performed to identify factors associated with mortality. Results As of 22 May 2020, 4393 patients were included, of whom 419 (9.5%) were HCW. Median (interquartile range) age of HCW was 52 (15) years and 62.4% were women. Prevalence of comorbidities and severe radiological findings upon admission were less frequent in HCW. There were no difference in need of respiratory support and admission to intensive care unit, but occurrence of sepsis and in-hospital mortality was lower in HCW (1.7% vs. 3.9%; p = 0.024 and 0.7% vs. 4.8%; p<0.001 respectively). Age, male sex and comorbidity, were independently associated with higher in-hospital mortality and healthcare working with lower mortality (OR 0.211, 95%CI 0.067-0.667, p = 0.008). 30-days survival was higher in HCW (0.968 vs. 0.851 p<0.001). Conclusions Hospitalized COVID-19 HCW had fewer comorbidities and a better prognosis than NHCW. Our results suggest that professional exposure to COVID-19 in HCW does not carry more clinical severity nor mortality

Systemic therapy for cervical cancer with potentially regulatable oncolytic adenoviruses

Peer reviewe

Visualization of primary tumor in the spleen and liver metastasis.

<p>Laparotomy observations of the entire organs and sections of the organs where the tumors are engrafted give an accurate idea of the location and shape of the spleen and liver tumors within the organ, which corresponds to its predicted location and shape by MRI.</p

Developmental stage of the spleens of adult SCID and NMRI nude mice.

<p>125 to 135 days old SCID and NMRI nude mice from different providers were imaged with MRI to evaluate the developmental stage of the spleens (arrows). For SCID mice, animals purchased from Charles River (A) and Harlan (B) showed poorly developed, diffuse, and small spleens while animals from Taconic (C) had solid, compact, and relatively large spleens. NMRI nude mice from Charles River (D) and Scanbur (E) showed fully developed spleens with a similar appearance.</p