In biomedical research, modes-of-action of modern targeted compounds are generally predicted by complex computer-assisted in silico modeling, followed by cell-biological validation and high-resolution imaging procedures to visualize compound interaction with respective molecular targets [
35,
36,
37,
38]. However, an additional factor impacting on compound efficacy is defined by its intracellular behavior. Knowledge of the intracellular dynamics of anticancer compounds is crucial, as it becomes increasingly evident that - besides known adaptive mechanisms such as target mutation, activation of alternative oncogenic pathways or transmembrane-transporter-mediated drug efflux - also other factors such as selective compound sequestration to cellular organelles have an impact on compound efficacy and can result in cellular unresponsiveness [
20,
21,
39]. In general, knowledge of intracellular pharmacokinetics is limited due to the highly complex nature of technologies capable of resolving compounds at subcellular level. These include elegant technologies such as hyperspectral Raman scattering spectroscopy [
30], nanoscale secondary ion mass spectrometry (nanoSIMS [
40]) or diaminobenzidine (DAB) photo-oxidation [
41]. The latter two approaches combine ultrastructural resolution methods with analytical chemistry. However, these technologies are restricted to the detection of metal-containing compounds (nanoSIMS) or to the physicochemical ability of compounds to generate reactive oxygen species upon excitation with light of specific wavelengths (DAB photo-oxidation [
41,
42]). Alternatively, artificial drug labeling represents a feasible strategy for intracellular tracking which, however, bears the risk of altering the pharmacological behavior of the respective compound [
43]. Intrinsic fluorescence activity has been described for only a limited number of chemotherapeutic and targeted agents including doxorubicin, mitoxantrone, topotecan, gefitinib, erlotinib, sunitinib and nintedanib [
16,
17,
18,
19,
20]. Nevertheless, exploitation of these fluorescence properties to study the intracellular compound dynamics has generated substantial advancements in the understanding of their modes of action and - importantly - also their limitations and failure [
19,
23,
30,
39]. As such, ion-based sequestration of widely used therapeutic agents such as doxorubicin, mitoxantrone, sunitinib, but also the FGFR inhibitor nintedanib to lysosomes was identified as a cancer cell defense mechanism [
16,
19,
20,
44]. These means to reduce cytoplasmic levels of pharmacologic agents was described to be further fostered by increased transcription factor EB (TFEB)-orchestrated lysosomal biogenesis and - as shown for topotecan, sunitinib and doxorubicin - by exocytosis of intralysosomal contents [
22]. At least in the case of nintedanib, which is clinically approved for the treatment of non-small cell lung cancer, concomitant targeting of lysosomes was shown to potentiate its activity against lung cancer cell lines in vitro [
20]. This goes well in accordance with the here-presented data for PD173074, especially because nintedanib is also considered a lipophilic (logP at pH 7.4 = 3.0), weakly basic compound, with a pKa of 7.9, based on the presence of a piperazine moiety exhibiting the potential for protonation. As mentioned above, another clinically approved tyrosine kinase inhibitor, imatinib, has been shown to accumulate in lysosomes [
30]. Also in this case, a pKa (8.1) comparable to that of nintedanib (due to the presence of a piperazine moiety in both molecules) reflects compound properties making lysosomotropism likely. It is tempting to ask whether chemical modification of anticancer compounds to reduce lysosomotropism might enhance their cytotoxic potential. Extensive research efforts would be necessary to identify derivatives exhibiting optimized intracellular pharmacokinetics, reduced organelle sequestration and, at the same time, sustained anticancer activity. This might prove challenging in the light of the fact that minor modifications in the chemical make-up of a compound are likely to strongly affect its physicochemical properties (including lipophilicity and the tendency to become protonated) and, thus, its pharmacological behavior. Consequently, it is conceivable that optimization of therapy containing lysosomotropic agents may best be achieved by rationale combination with agents targeting lysosomal integrity. Attempts have been launched to compromise the lysosomal integrity of cancer cells in order to increase the cytotoxic potential of lysosomotropic anticancer compounds. As mentioned, such approaches include pharmacological lysosome alkalinization via luminal proton scavenging (e.g., by chloroquine) or v-ATPase inhibition (e.g., by bafilomycin A1) [
45]. Other strategies employ pharmacological Trojan horses such as imidazoacridinones or so-called photoaccoustic nanobombs, which themselves exhibit lysosomotropic properties. These have been developed for the photodynamic destruction of lysosomes to induce cancer cell death and might prove beneficial for combination with lysosomotropic anticancer compounds aimed at other cellular targets [
46,
47,
48].
In addition to lysosomes, other organelles are implicated in the scavenging of pharmacological agents. For instance, adiposomal accumulation of the preclinical compound curcumin was suggested to lower its anticancer activity [
49]. Another work described late endosomal sequestration of doxorubicin to mediate cancer cell resistance [
23]. Accordingly, these studies demonstrated that combination treatments with compounds compromising the physiological integrity of the above-mentioned cellular compartments yield synergistic anti-proliferative effects, presumably by elevating drug levels available to act at their respective target sites. In line with these observations, in our study we have identified lysosomal release and enhanced toxicity in FGFR-driven lung cancer cells by combination of PD173074 with compounds targeting lysosomal pH. This is most likely mediated by enhanced local drug concentrations at the intracellular side of the membrane to interact with the FGFR kinase domain.