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Tumor drug penetration measurement reveals limited distribution of fluor-conjugated Herceptin antibody in a mouse breast cancer model

One of the major challenges for cancer therapy is the occurrence of side effects.  The problem remains unresolved for either cytostatic or cytotoxic drugs.  Whereas cytostatic drugs arrest cell cycling, cytotoxic drugs induce cell death.  While conventional therapies like the chemotherapy or radiotherapy are cytotoxic, many of the 'molecularly targeted drugs' developed in the last several decades (that inhibit proteins expressed by oncogenes) tend to be cytostatic.  Oncogenes are derived from proto-oncogenes in our genome, which are thought to promote cancer upon acquiring mutation(s) that upregulates its activity (ex. tyrosine kinase).  This is the key reason (treated cells remain viable) why targeted drugs like Gleevec (for chronic myelogenous leukemia) need to be administered perennially to avoid recurrent cancer.  Yet, even with targeted drugs, the problem of side effects persists.  For Gleevec, the side effects include rash, infection, neutropenia, anemia, edema, thrombocytopenia, etc.

Another contributing factor to side effects is that a significant fraction of intravenously injected drugs fail to reach tumors.  In the case of Taxol, nearly half of all injected drugs are eliminated after 24 h, with less than 0.5% remaining locally to treat lung cancer (Wolinsky et al., 2012).  To compensate, a greater dose of drugs may need to be administered approaching the 'maximum tolerated dose' (MTD), further exacerbating side effects. 

Several methods have been developed to address the issue.  One such approach is chemoembolization (for liver cancer), wherein the artery feeding into liver is blocked off after administering the drugs to trap them inside the liver, thus avoiding them from circulating throughout the body.  However, the utility of this strategy is limited and may not be applicable for other types of cancer.

An alternate approach involves conjugating drugs to tumor targeting delivery vectors.  One such strategy sought to utilize antibodies to guide drugs to tumors.  Tumor targeting antibodies have been increasingly used for cancer therapy.  Among them is Herceptin, a monoclonal antibody that recognizes Her2 oncogene overexpressed in a subset (~15%) of breast cancers (Figueroa-Magalhães et al., 2014).


          
 

To assess its efficacy as a delivery vector, the ability of Herceptin to penetrate solid tumors was examined (solid tumors comprise >95% of human cancers).  To measure the depth of penetration, the investigators at the University of Chicago developed a novel imaging technique through which microscopic images taken at multiple depths could be integrated using computer to build the 3-dimensional 'map' of a tumor (Lee et al., 2019).  Then, by applying the principle of tomography (ex. CAT-SCAN imaging), the 3D map could be viewed at any axis (X- or Y- or Z-axis) to determine drug distribution within a solid tumor. 

To trace the injected drug, Herceptin antibody was conjugated to the fluor DyLight594.  DyLight fluors are fluorescent dyes activated at similar wavelengths as conventional fluorphores (ex. fluoresceine, Cy5, rhodamine) but may be more photostable (takes longer time to photobleach by laser). To measure the distance it travelled after leaving a blood vessel, they conjugated anti-CD31 antibody to DyLight633 fluorescent dye to locate blood vessels (for reference point).

Following the intravenous injection of both antibodies in a transgenic mouse model (overexpresses neu, a murine homologue of Her2) that spontaneously develops mammary tumors, they determined that Herceptin traveled mere 38 micrometer (several cell length) from the blood vessel boundaries after 1 hour-post-injection. 

In another report, a greater distribution of Herceptin was observed after 1 day-post-injection; however, the antibody still could not reach hypoxic regions of a solid tumor (Lee et al., 2010).  Hypoxic regions are located distant from blood vessels within a solid tumor and suffer from a low level of oxygen or nutrients, a higher mutation rate, and drug resistance (Sullivan et al., 2008; Li et al., 2017).  In this regard, delivery vectors capable of penetrating solid tumors deeply like the tumor-targeting peptides may play a greater role in reducing side effects (Joliot et al., 2004; Wright et al., 2016; Hong et al., 2000; Li et al., 2021).

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References

Figueroa-Magalhães MC, et al. Treatment of HER2-positive breast cancer.  Breast. 23:128-136 (2014). PMID: 24360619

Hong FD, Clayman GL. Isolation of a peptide for targeted drug delivery into human head and neck solid tumors.  Cancer Res. 2000 Dec 1;60(23):6551-6.   PMID: 11118031

Joliot A, Prochiantz A. Transduction peptides: from technology to physiology.   Nat Cell Biol.  6:189-96 (2004).  PMID: 15039791

Lee CM, Tannock IF. The distribution of the therapeutic monoclonal antibodies cetuximab and trastuzumab within solid tumors.  BMC Cancer.  10:255 (2010).  PMID: 20525277

Lee SS, Bindokas VP, et al. Multiplex Three-Dimensional Mapping of Macromolecular Drug Distribution in the Tumor Microenvironment.  Mol Cancer Ther.  18:213-226 (2019).  PMID: 30322947

Li JQ, Wu X, Gan L, et al.   Hypoxia induces universal but differential drug resistance and impairs anticancer mechanisms of 5-fluorouracil in hepatoma cells.   Acta Pharmacol Sin. 38:1642-1654 (2017). PMID: 28713155

Li R, Wang Y, et al. Graphene oxide loaded with tumor-targeted peptide and anti-cancer drugs for cancer target therapy.  Sci Rep. 11:1725 (2021).  PMID: 33462277

Sullivan R, Paré GC, et al.  Hypoxia-induced resistance to anticancer drugs is associated with decreased senescence and requires hypoxia-inducible factor-1 activity.  Mol Cancer Ther.  7:1961-73 (2008).  PMID: 18645006

Wolinsky JB, Colson YL, et al. Local drug delivery strategies for cancer treatment: gels, nanoparticles, polymeric films, rods, and wafers.   J Control Release. 159:14-26 (2012).   PMID: 22154931

Wright CL, Pan Q, . Advancing theranostics with tumor-targeting peptides for precision otolaryngology.   World J Otorhinolaryngol Head Neck Surg.  2:98-108 (2016).  PMID: 29204554